Composition and method for muscle repair and regeneration

ABSTRACT

The invention provides methods for muscle repair or regeneration comprising administering therapeutically effective amounts of RAR agonists or stem cells that are pretreated with contact with a RAR agonist to a subject at a site of muscle damage. Additionally, the invention provides compositions comprising RAR agonist treated stem cells and methods of use of said cells for muscle repair or regeneration. In one embodiment, the stem cells are mesenchymal stem cells. In one embodiment, the RAR agonist is an RARγ agonist. In one embodiment, administration of the RAR agonist is begun during a period of increased endogenous retinoid signaling in the subject resulting from incurrence of the damaged muscle tissue.

RELATED APPLICATIONS

This application claims the benefit of 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 61/378,996, filed Sep. 1, 2010, thecontents of which are incorporated herein by reference in theirentirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.W81XWH-07-1-0212 awarded by the United States Army. The government hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for musclerepair and regeneration.

BACKGROUND OF THE INVENTION

Injuries to muscles include acute injuries to skeletal muscles such ascontusions (bruises), lacerations, ischemia, strains, and completeruptures. These injuries may cause tremendous pain and can incapacitatethe affected person, preventing them from being able to go to work oreven to participate in normal daily activities.

Skeletal muscles can become injured for a variety of reasons includingover-use, trauma, infections and loss of blood circulation. In general,muscles have adequate repair capacity particularly in young people, butthis repair process can become ineffective in older people or afterrepeated rounds of over-use, severe trauma or other processes. In suchcases, the muscles lose function and strength of contraction and can bereplaced by scar tissue (connective tissue) that by lackingcontractility, causes loss of muscle function. Current therapies includemassage, ultrasound, hyperbaric oxygen delivery, and drug treatmentssuch as fenoterol and insulin-like growth factor-1. These therapies canprovide some beneficial outcomes, but are far from ideal in terms ofeffectiveness and efficiency. In addition, current therapies are notspecifically directed toward basic mechanisms of muscle cell repair andregeneration.

Accordingly, there exists a need for methods and compositions that caninduce muscle repair and regeneration speedily and effectively.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for muscle repair orregeneration in a subject, the method comprising administering atherapeutically effective amount of a retinoic acid receptor (RAR)agonist to a subject, which subject has a damaged muscle tissue. In someembodiments, the RAR agonist is a RARγ agonist.

One aspect of the invention relates to a method of muscle repair orregeneration in a subject, comprising administering a therapeuticallyeffective amount of a retinoic acid receptor gamma (RARγ) agonist to asubject with damaged muscle tissue, to thereby repair or regenerate thedamaged muscle tissue. In one embodiment of the methods describedherein, the administration is local or systemic. In one embodiment ofthe methods described herein, administration is begun during a timeperiod of increased endogenous retinoid signaling in the subjectresulting from incurrence of the damaged muscle tissue. In oneembodiment of the methods described herein, administration is begunlater than 3 days after incurrence of the damaged muscle tissue by thesubject. In one embodiment of the methods described herein,administration is begun at about 4 days after incurrence of the damagedmuscle tissue by the subject. In one embodiment of the methods describedherein, administration is begun at about 5 days after incurrence of thedamaged muscle tissue by the subject. In one embodiment of the methodsdescribed herein, administration is begun at about 6 days afterincurrence of the damaged muscle tissue by the subject. In oneembodiment of the methods described herein, administration is begun atabout 7 days after incurrence of the damaged muscle tissue by thesubject. In one embodiment of the methods described herein,administration is begun at about 5 days, and is continued through to atleast day 7 after incurrence of the damaged muscle tissue by thesubject. In one embodiment of the methods described herein,administration is continued through to at least day 9 after incurrenceof the damaged muscle tissue by the subject. In one embodiment of themethods described herein, the RARγ agonist is selected from the groupconsisting of CD-271(6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid); CD-394, CD-437(6-(4-Hydroxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid); CD-1530(4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoicacid); CD-2247; palovarotene(4-[(1E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]-benzoic acid); BMS-270394(3-Fluoro-4-[(R)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic acid); BMS-189961(3-Fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoicacid); CH-55(4-[(E)-3-(3,5-Di-tert-butyl-phenyl)-3-oxo-propenyl]-benzoic acid);6-[3-(adamantan-1-yl)-4-(prop-2-ynyloxy)phenyl]naphthalene-2-carboxylicacid;5-[(E)-3-oxo-3-(5,5,8,8-tetrahydronaphthalene-2-yl)propenyl]thiophene-2-carboxylicacid; and enantiomers, derivatives, prodrugs, and pharmaceuticallyacceptable salts thereof. In one embodiment of the methods describedherein, the damaged muscle tissue is the result of a physical injury oraccident, disease, infection, over-use, loss of blood circulation, ormuscle atrophy or wasting. In one embodiment of the methods describedherein, the damaged muscle tissue is dystrophic muscle or an ageingmuscle. In one embodiment of the methods described herein, the damagedmuscle tissue is the result of muscle atrophy/wasting. In one embodimentof the methods described herein, the subject is a mammal. In oneembodiment of the methods described herein, the subject is a mouse. Inone embodiment of the methods described herein, the subject is human. Inone embodiment of the methods described herein, the method furthercomprises administering an anti-inflammatory agent to the subject.

Another aspect of the present invention relates to a method of musclerepair or regeneration in a subject, comprising administeringpluripotent or multipotent stem cells that have been pre-treated with aRARγ agonist, to a subject at a site of muscle injury, to thereby repairor regenerate muscle at the site. In one embodiment of the methodsdescribed herein, the muscle injury is a compound tissue injury. In oneembodiment of the methods described herein the compound tissue injurycomprises an injury to muscle and bone. In one embodiment of the methodsdescribed herein the pre-treated stem cells are administered incombination with non-treated stem cells. In one embodiment of themethods described herein the pre-treated stem cells and non-treated stemcells are administered at a ratio of 1:1. In one embodiment of themethods described herein, the pre-treated stem cells have beenpre-treated with the RARγ agonist for a period of about 3 days. In oneembodiment of the methods described herein, the pluripotent stem cellsare induced pluripotent stem cells. In one embodiment of the methodsdescribed herein, the pluripotent stem cells are mesenchymal stem cells.In one embodiment of the methods described herein, administering islocal. In one embodiment of the methods described herein, administeringis by transplantation of the cells into the subject. In one embodimentof the methods described herein, the pluripotent stem cells areautologous or heterologous to the subject. In one embodiment of themethods described herein, the pluripotent stem cells are mammalian. Inone embodiment of the methods described herein, the pluripotent stemcells are rodent. In one embodiment of the methods described herein, thepluripotent stem cells are human. In one embodiment of the methodsdescribed herein, the method further comprises administering ananti-inflammatory agent to the subject. In one embodiment of the methodsdescribed herein, the RAR agonist is selected from the group consistingof CD-271(6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid); CD-394, CD-437(6-(4-Hydroxy-3-ricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid); CD-1530(4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoicacid); CD-2247; palovarotene(4-[(1E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]-benzoicacid); BMS-270394(3-Fluoro-4-[(R)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoicacid); BMS-189961(3-Fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoicacid); CH-55(4-[(E)-3-(3,5-Di-tert-butyl-phenyl)-3-oxo-propenyl]-benzoic acid);6-[3-(adamantan-1-yl)-4-(prop-2-ynyloxy)phenyl]naphthalene-2-carboxylicacid;5-[(E)-3-oxo-3-(5,5,8,8-tetrahydronaphthalene-2-yl)propenyl]thiophene-2-carboxylicacid; and enantiomers, derivatives, prodrugs, pharmaceuticallyacceptable salts thereof, and combinations thereof. In one embodiment ofthe methods described herein, the injured muscle results from physicalinjury or accident, disease, infection, over-use, loss of bloodcirculation, or muscle atrophy or wasting. In one embodiment of themethods described herein, the injured muscle tissue is dystrophic muscleor an ageing muscle. In one embodiment of the methods described herein,the injured muscle is the result of muscle atrophy/wasting.

Another aspect of the invention relates to a method of inducing orstimulating myogenic differentiation of isolated mesencymal stem cellsin vitro comprising, contacting the mesencymal stem cells with aneffective amount of a retinoic acid receptor gamma (RARγ) agonist. Inone embodiment of the methods described herein, contacting is for a timeperiod selected from the group consisting of about 12 hours, about 1day, about 2 days, and about 3 days.

Another aspect of the invention relates to a method of inducing orstimulating lineage-directed differentiation of a pluripotent stem cellinto a mesenchymal lineage, the method comprising contacting thepluripotent stem cell with an effective amount of a retinoic acidreceptor gamma (RARγ) agonist. In one embodiment of the methodsdescribed herein, the pluripotent stem cell is an induced pluripotentstem cell. In one embodiment of the methods described herein, thepluripotent stem cell is a mesenchymal stem cell. In one embodiment ofthe methods described herein, the mesenchymal lineage is selected fromthe group consisting of myogenic, osteogenic, chodrogenic, tendonogenic,ligamentogenic, maroow stromagenic, adipogenic, and dermogenic. In oneembodiment of the methods described herein, the pluripotent stem cell ismammalian. In one embodiment of the methods described herein, thepluripotent stem cell is rodent. In one embodiment of the methodsdescribed herein, the pluripotent stem cell is human. In one embodimentof the methods described herein, the RARγ agonist is selected from thegroup consisting of CD-271(6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid); CD-394. CD-437(6-(4-Hydroxy-3-tricyclo[3.3.1.13.7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid); CD-1530(4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoicacid); CD-2247; palovarotene(4-[(1E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]-benzoicacid); BMS-270394(3-Fluoro-4-[(R)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoicacid); BMS-189961(3-Fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoicacid); CH-55(4-[(E)-3-(3,5-Di-tert-butyl-phenyl)-3-oxo-propenyl]-benzoic acid);6-[3-(adamantan-1-yl)-4-(prop-2-ynyloxy)phenyl]naphthalene-2-carboxylicacid;5-[(E)-3-oxo-3-(5,5,8,8-tetrahydronaphthalene-2-yl)propenyl]thiophene-2-carboxylicacid; and enantiomers, derivatives, prodrugs, pharmaceuticallyacceptable salts thereof, and combinations thereof.

Another aspect of the invention relates to a composition comprising amesenchymal stem cells wherein a portion of the mesenchymal stem cellshave been pretreated by contact with a RARγ agonist to thereby generatepretreated mesenchymal stem cells. In one embodiment of the compositionsdescribed herein, the portion of pretreated mesenchymal stem cells isselected from the group consisting of 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, and 90%.

Another aspect of the invention relates to a composition comprising apluripotent stem cell population, wherein at least one cell of thepopulation was contacted with a RARγ agonist to thereby generate apretreated pluripotent stem cell. In one embodiment of the compositionsdescribed herein, the pluripotent stem cell is an induced pluripotentcell. In one embodiment of the compositions described herein, thepluripotent stem cell is a mesenchymal stem cell. In one embodiment ofthe compositions described herein, the stem cell is an isolated stemcell. In one embodiment of the compositions described herein, the stemcell is mammalian. In one embodiment of the compositions describedherein, the stem cell is murine. In one embodiment of the compositionsdescribed herein, the stem cell is human. In one embodiment of thecompositions described herein, the RARγ agonist is selected from thegroup consisting of CD-271(6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid); CD-394, CD-437(6-(4-Hydroxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid); CD-1530(4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoicacid); CD-2247; palovarotene(4-[(E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]-benzoicacid); BMS-270394(3-Fluoro-4-[(R)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoicacid); BMS-189961(3-Fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoicacid); CH-55(4-[(E)-3-(3,5-Di-tert-butyl-phenyl)-3-oxo-propenyl]-benzoic acid);6-[3-(adamantan-1-yl)-4-(prop-2-ynyloxy)phenyl]naphthalene-2-carboxylicacid;5-[(E)-3-oxo-3-(5,5,8,8-tetrahydronaphthalene-2-yl)propenyl]thiophene-2-carboxylicacid; and enantiomers, derivatives, prodrugs, pharmaceuticallyacceptable salts thereof, and combinations thereof.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising a composition comprising a pluripotent stem cellpopulations described herein and a pharmaceutically acceptable carrier.

Another aspect of the present invention relates to a kit for repairingor regenerating muscle, comprising at least one of a RARγ agonist, aRARγ agonist and a stem cell, or a composition comprising a stem cell,described herein. In one embodiment of the kits described herein, thestem cell is an induced pluripotent stem cell. In one embodiment of thekits described herein, the stem cell is a mesenchymal stem cell. In oneembodiment of the kits described herein, the RARγ agonist is formulatedin a pharmaceutical composition. In one embodiment of the kits describedherein, the RARγ agonist is formulated for topical application. In oneembodiment of the kits described herein, the stem cell is an isolatedstem cell. In one embodiment of the kits described herein, the stem cellis mammalian. In one embodiment of the kits described herein, the stemcell is rodent. In one embodiment of the kits described herein, the stemcell is human. In one embodiment of the kits described herein, the RARγagonist is selected from the group consisting of CD-271(6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid); CD-394, CD-437(6-(4-Hydroxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid); CD-1530(4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoicacid); CD-2247; palovarotene(4-[(1E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]-benzoicacid); BMS-270394(3-Fluoro-4-[(R)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic acid); BMS-189961(3-Fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoicacid); CH-55(4-[(E)-3-(3,5-Di-tert-butyl-phenyl)-3-oxo-propenyl]-benzoic acid);6-[3-(adamantan-1-yl)-4-(prop-2-ynyloxy)phenyl]naphthalene-2-carboxylicacid;5-[(E)-3-oxo-3-(5,5,8,8-tetrahydronaphthalene-2-yl)propenyl]thiophene-2-carboxylicacid; and enantiomers, derivatives, prodrugs, pharmaceuticallyacceptable salts thereof, and combinations thereof. In one embodiment ofthe kits described herein, the kit further comprises instructions foruse.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph of data obtained from the histological analysis ofskeletal muscle tissue 4-week after injury. To evaluate and quantify thechanges of skeletal muscle tissue structure, images of trichrome-stainedserial sections were taken and used to determine the relative amounts ofmuscle fiber, adipose and fibrous tissues in multiple defined areas byImagePro software. The analyzed areas were 9×9 grids each (about 3×3 mm)that included the original injured site (2×2 mm). Note that muscleinjury caused a decrease in total muscle fiber area and a concurrentincrease in fibrous and adipose tissues area. In contrast, CD1530treatment largely restored the tissue composition.

FIG. 2 is a graphical representation of data obtained fromhisto-morphometric analysis of the composition of injured muscle.

FIG. 3 are names and chemical structures of some exemplary RARγagonists.

FIG. 4 is a series of bar graphs with data from experimental resultsthat indicate the local RA concentration decreases first and thentransiently increases after muscle injury.

FIG. 5 is a series of line graphs with data from experimental resultsthat indicate the local RA concentration decreases first and thentransiently increases after muscle injury.

FIG. 6 is a bar graph of data obtained from the histological analysis ofskeletal muscle tissue 4-week after injury following the indicatedtreatment times with RARγ agonist. On the graph, the left filled insegment indicates muscle. The segment to the direct right of that isshown as open (white) and indicates adipose. The segment to the right ofthat, filled in, indicates fibrous tissue.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the invention relate to the finding that progenitor stemcells can be induced to undergo muscle differentiation by acute orchronic exposure to retinoic acid receptor gamma (RARγ) agonists.Accordingly, in one aspect, the invention provides a method forrepairing or regenerating a damaged muscle tissue of a subject, themethod comprising administering a therapeutically effective amount of aretinoic acid receptor (RAR) agonist to a subject, which subject has adamaged muscle tissue.

As used herein, the term “damaged muscle tissue” refers to a muscletissue, such as a skeletal or cardiac muscle that has been altered forinstance by a physical injury or accident, disease, infection, over-use,loss of blood circulation, or by genetic or environmental factors. Adamaged muscle tissue can be a dystrophic muscle or an ageing muscle.Exemplary symptoms of muscle damage include, but are not limited to,swelling, bruising or redness, open cuts as a consequence of an injury,pain at rest, pain when specific muscle or the joint in relation to thatmuscle is used, weakness of the muscle or tendons, and an inability touse the muscle at all.

In some embodiments of this and other aspects of the invention, thedamaged muscle tissue results from muscle atrophy/wasting. In someembodiments of this and other aspects of the invention, the damagedmuscle tissue results from a physical injury.

In some embodiments of this and other aspects of the invention, thedamaged muscle is skeletal muscle.

In some embodiments of this and other aspects of the invention, diseaseresulting in damaged muscle tissue is a myopathy. Without limitation,myopathy can be a congenital myopathy or an acquired myopathy. Exemplarymyopathies include, but are not limited to, dystrophies, myotonia(neuromytonia), congenital myopathies (e.g., nemaline myopathy,multi/minicore myopathy, centronuclear myopathy (or myotubularmyopathy)), mitochondrial myopathies, familial periodic paralysis,inflammatory myopathies, metabolic myopathies (e.g., glycogen storagedisease and lipid storage disorder), dermatomyositis, polymyositisinclusion body myositis, myositis ossificans, rhabdomyolysis andmyoglobinuirias.

In some embodiments of this and other aspects of the invention, myopathyis a dystrophy selected from the group consisting of muscular dystrophy,Duchenne muscular dystrophy, Becker's muscular dystrophy, Reflexsympathetic dystrophy, Retinal dystrophy, Conal dystrophy, Myotonicdystrophy, Corneal dystrophy, and any combinations thereof.

Without wishing to be bound by a theory, methods described herein reduceand/or inhibit formation of scar-like tissue in the damaged muscletissue. Accordingly, in some embodiments, formation of scar-like tissueformation in the damaged muscle tissue is reduced by at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, or100% (complete reduction) relative to a reference level.

In some embodiments, amount of adipose tissue and/or connective tissuein the damaged muscle tissue is reduced by at least 5%, at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or 100% (completerreduction) relative to a reference level.

Again, without wishing to be bound by a theory, the methods describedherein lead to an increase in the amount of myosin heavy chain (MHC) inthe damaged muscle tissue. Accordingly, in some embodiments amount ofmyosin heavy chain (MHC) in the damaged muscle tissue in increased by atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, or100-fold or more, relative to reference level.

Additionally, the methods described herein increase the number of MHCpositive elongated muscle-like cells. Accordingly, in some embodiments,number of MHC positive elongated muscle-like cells in the damaged muscletissue is increased by at least 5%, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold,10-fold, 50-fold, or 100-fold or more, relative to reference level.

As discussed herein, progenitor stem cells can be induced or stimulatedto undergo muscle differentiation by acute or chronic exposure toretinoic acid receptor gamma (RARγ) agonists. Accordingly, an increasein myogenic regulatory factors is observed. Myogenic regulatory factorsare basic-helix-loop-helix (bHLH) transcription factors that regulatemyogenesis. See for example, Perry, R. & Rudnick, M. (2000). “Molecularmechanisms regulating myogenic determination and differentiation”, FrontBiosci 5: D750-67 (2000), content of which is herein incorporated byreference. Exemplary myogenic regulatory factors include, but are notlimited to, MyoD (Myf3), Myf5, myogenin, and MRF4 (Myf6). MyoD is one ofthe earliest markers of myogenic commitment. MyoD is expressed inactivated satellite cells, but not in quiescent satellite cells.Although MyoD marks myoblast commitment, muscle development is notdramatically ablated in mouse mutants lacking the MyoD gene. This islikely to be due to functional redundancy from Myf5.

Accordingly, in some embodiments, level of at least one myogenicregulatory factor in the damaged muscle tissue increases by at least 5%,at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least, I-fold,2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, or 100-fold or more,relative to reference level.

In some embodiments, amount of at least one laminin in the damagedmuscle tissue is increased by at least 5%, at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least, 1-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 50-fold, or 100-fold or more, relative to referencelevel.

Retinoic Acid Receptor (RAR) Agonists

As used herein, the term “RAR agonist” is any compound that is capableof transactivating any of the retinoic acid receptor with an ED50 ofless than 1000 nM, less than 500 nM, less than 250 nM, less than 200 nM,less than 100 nM, less than 50 nM, less than 25 nM, less than 20 nM,less than 10 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, orless than 0.001 nM.

The retinoid receptors are classified into two families, the retinoicacid receptors (RARs) and the retinoid X receptors (RXRs), eachconsisting of three distinct subtypes α, β, and γ. Each subtype of theRAR gene family encodes a variable number of isoforms arising fromdifferential splicing of two primary RNA transcripts. All-trans retinoicacid (ATRA) and its other naturally occurring retinoid analogs (9-cisretinioc acid, all-trans 3-4 didehydro retinioc acid, 4-oxo retinoicacid and retinol) are pleiotrophic regulatory compounds that bind withretinoid receptors. For example, ATRA binds with approximately equalaffinity to all the three RAR subtypesm, but does not bind to the RXRreceptors. Instead, for these receptors, 9-cis retinoic acid is thenatural ligand.

As used herein, the term “transactivation” refers to the ability of anRAR agonist to activate the transcription of a gene where the genetranscription is initiated by the binding of a ligand to the particularretinoic acid receptor being tested, i.e., RARα, RARβ, or RARγ.Determining the ability of a compound to transactivate a retinoic acidreceptor may be performed by methods known to those of skill in the art.Examples of such methods are found in Bernard et al., Biochem. Biophys.Res. Commun., 186: 977-983 (1992) and Apfel et al., Proc. Nat. Sci.Acad. (USA), 89: 7129-7133 (1992), content of both of which is hereinincorporated by reference.

In some embodiments of the aspects described herein, the RAR agonist isa RARγ selective agonist. As used herein, the term “RARγ selectiveagonist” refers to a compound that is able to selectively bind to theRARγ receptor and promote RARγ activation. Generally, RARγ selectiveagonists will bind to the RARγ receptor at significantly lowerconcentrations than the RARα and RARβ receptors. For example, a RARγselective agonist will bind to the RARγ receptor with a more than5-fold, more than 10-fold, more than 20-fold, more than 30-fold, morethan 40-fold, more than 50-fold, more than 60-fold, more than 70-fold,more than 80-fold, more than 90-fold or more selectivity, than the RARαand RARβ receptors.

The RAR agonist selectivity of a compound can be determined by routineligand binding assays known to one of skill in the art such as describedin Apfel et al., Proc. Nat. Sci. Acad. (USA), 89: 7129-7133 (1992); M.Teng et al., J. Med. Chem., 40: 2445-2451 (1997); and PCT Pub. No.WO1996/30009, content of all of which is herein incorporated byreference.

In some embodiments of the aspects described herein, the RAR agonist isa RARγ/β selective agonist. As used herein, the term “RARγ/β selectiveagonist” refers to a compound that selectively binds to RARγ and RARβreceptors, promoting both RARγ and RARβ activation and sparing theactivation of RARα receptors.

In some embodiments of the aspects described herein, the RAR agonist isa RAR agonist that is at least gamma selective and is RARα sparing. Asused herein, the term “RAR agonist that is at least gamma selective andis RARα sparing” refers to a compound that is RARγ selective or RARγ/βselective.

In some embodiments of the aspects described herein, the RAR agonist isa RAR pan agonist. As used herein, the term “RAR pan agonist” refers toa compound that binds to RARα, RARβ, and RARγ receptors with similaraffinity, promoting RARα, RARβ, and RARγ activation.

Exemplary, RAR agonists include, but are not limited to, CD-271(6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid); CD-394, CD-437(6-(4-Hydroxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid); CD-1530(4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoicacid); CD-2247; palovarotene(4-[(1E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]-benzoicacid); BMS-270394(3-Fluoro-4-[(R)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoicacid); BMS-189961(3-Fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoicacid); CH-55(4-[(E)-3-(3,5-Di-tert-butyl-phenyl)-3-oxo-propenyl]-benzoic acid);6-[3-(adamantan-1-yl)-4-(prop-2-ynyloxy)phenyl]naphthalene-2-carboxylicacid;5-[(E)-3-oxo-3-(5,5,8,8-tetrahydronaphthalene-2-yl)propenyl]thiophene-2-carboxylicacid; and enantiomers, derivatives, prodrugs, and pharmaceuticallyacceptable salts thereof.

Other RAR agonists amenable to the present invention are described, forexample, in U.S. Pat. Nos. 5,624,957; 5,760,084; 6,331,570; 6,300,350;5,700,836; 5,726,191; 5,498,795; 5,130,335; Int. Pat. App. Pub. No.WO1997/037648, No. WO2007/068579, and No. WO2007/068580; French Pat.App. No. FR2739557 published Apr. 11, 1997; and Japanese Pat. Pub. No.62/053981, content of all which is herein incorporated by reference inits entirety. Further RAR agonists amenable to the present inventioninclude those described in Biochem. Biophys. Res. Commun. 179: 1554-1561(1992), Biochem. Biophys. Res. Commun. 186: 977-984 (1992), Int. J.Cancer 71: 497 (1997), Skin Pharmacol. 8: 292-299 (1995). J. Med. Chem.39: 2411-2421 (1996). Cancer Res. 55: 4446-4451 (1995), Cancer Letters115: 1-7 (1997), J. Med. Chem. 32: 834-840 (1989), content of all ofwhich is herein incorporated by reference.

Pharmaceutically Acceptable Salts

As used herein, the term “pharmaceutically-acceptable salts” refers tothe conventional nontoxic salts or quaternary ammonium salts of the RARagonists, e.g., from non-toxic organic or inorganic acids. These saltscan be prepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting a purified RAR agonistin its free base or acid form with a suitable organic or inorganic acidor base, and isolating the salt thus formed during subsequentpurification. Conventional nontoxic salts include those derived frominorganic acids such as sulfuric, sulfamic, phosphoric, nitric, and thelike; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic,and the like. See, for example. Berge et al., “Pharmaceutical Salts”, J.Pharm. Sci. 66:1-19 (1977), content of which is herein incorporated byreference in its entirety.

In some embodiments of the aspects described herein, representativesalts include the hydrobromide, hydrochloride, sulfate, bisulfate,phosphate, nitrate, acetate, succinate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.

Prodrugs

As used herein, a “prodrug” refers to compounds that can be convertedvia some chemical or physiological process (e.g., enzymatic processesand metabolic hydrolysis) to a RAR agonist. Thus, the term “prodrug”also refers to a precursor of a biologically active compound that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject, i.e. an ester, but is converted in vivo to an activecompound, for example, by hydrolysis to the free carboxylic acid or freehydroxyl. The prodrug compound often offers advantages of solubility,tissue compatibility or delayed release in an organism. The term“prodrug” is also meant to include any covalently bonded carriers, whichrelease the active compound in vivo when such prodrug is administered toa subject. Prodrugs of an active compound may be prepared by modifyingfunctional groups present in the active compound in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent active compound. Prodrugs include compounds wherein ahydroxy, amino or mercapto group is bonded to any group that, when theprodrug of the active compound is administered to a subject, cleaves toform a free hydroxy, free amino or free mercapto group, respectively.Examples of prodrugs include, but are not limited to, acetate, formateand benzoate derivatives of an alcohol or acetamide, formamide andbenzamide derivatives of an amine functional group in the activecompound and the like. See Harper, “Drug Latentiation” in Jucker, ed.Progress in Drug Research 4:221-294 (1962); Morozowich et al,“Application of Physical Organic Principles to Prodrug Design” in E. B.Roche ed. Design of Biopharmaceutical Properties through Prodrugs andAnalogs, APHA Acad. Pharm. Sci. 40 (1977); Bioreversible Carriers inDrug in Drug Design, Theory and Application, E. B. Roche, ed., APHAAcad. Pharm. Sci. (1987); Design of Prodrugs, H. Bundgaard, Elsevier(1985); Wang et al. “Prodrug approaches to the improved delivery ofpeptide drug” in Curr. Pharm. Design. 5(4):265-287 (1999); Pauletti etal. (1997) Improvement in peptide bioavailability: Peptidomimetics andProdrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al.(1998) “The Use of Esters as Prodrugs for Oral Delivery of (3-Lactamantibiotics,” Pharm. Biotech. 11:345-365; Gaignault et al. (1996)“Designing Prodrugs and Bioprecursors I. Carrier Prodrugs,” Pract. Med.Chem. 671-696; Asgharnejad, “Improving Oral Drug Transport”, inTransport Processes in Pharmaceutical Systems, G. L. Amidon, P. I. Leeand E. M. Topp, Eds. Marcell Dekker, p. 185-218 (2000); Balant et al.,“Prodrugs for the improvement of drug absorption via different routes ofadministration”, Eur. J. Drug Metab. Pharmacokinet., 15(2): 143-53(1990): Balimane and Sinko, “Involvement of multiple transporters in theoral absorption of nucleoside analogues”, Adv. Drug Delivery Rev.,39(1-3): 183-209 (1999); Browne, “Fosphenytoin (Cerebyx)”, Clin.Neuropharmacol. 20(1): 1-12 (1997); Bundgaard, “Bioreversiblederivatization of drugs-principle and applicability to improve thetherapeutic effects of drugs”, Arch. Pharm. Chemi 86(1): 1-39 (1979);Bundgaard H. “Improved drug delivery by the prodrug approach”,Controlled Drug Delivery 17: 179-96 (1987); Bundgaard H. “Prodrugs as ameans to improve the delivery of peptide drugs”, Arfv. Drug DeliveryRev. 8(1): 1-38 (1992); Fleisher et al. “Improved oral drug delivery:solubility limitations overcome by the use of prodrugs”, Arfv. DrugDelivery Rev. 19(2): 115-130 (1996); Fleisher et al. “Design of prodrugsfor improved gastrointestinal absorption by intestinal enzymetargeting”, Methods Enzymol. 112 (Drug Enzyme Targeting, Pt. A): 360-81,(1985); Farquhar D, et al., “Biologically ReversiblePhosphate-Protective Groups”, Pharm. Sci., 72(3): 324-325 (1983);Freeman S. et al., “Bioreversible Protection for the Phospho Group:Chemical Stability and Bioactivation of Di(4-acetoxy-benzyl)Methylphosphonate with Carboxyesterase,” Chem. Soc., Chem. Commun.,875-877 (1991); Friis and Bundgaard, “Prodrugs of phosphates andphosphonates; Novel lipophilic alphaacyloxyalkyl ester derivatives ofphosphate- or phosphonate containing drugs masking the negative chargesof these groups”, Eur. J. Pharm. Sci. 4: 49-59 (1996); Gangwar et al.,“Pro-drug, molecular structure and percutaneous delivery”, Des.Biopharm. Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409-21.(1977); Nathwani and Wood, “Penicillins: a current review of theirclinical pharmacology and therapeutic use”, Drugs 45(6): 866-94 (1993);Sinhababu and Thakker, “Prodrugs of anticancer agents”, Adv. DrugDelivery Rev. 19(2): 241-273 (1996); Stella et al., “Prodrugs. Do theyhave advantages in clinical practice?”, Drugs 29(5): 455-73 (1985); Tanet al. “Development and optimization of anti-HIV nucleoside analogs andprodrugs: A review of their cellular pharmacology, structure-activityrelationships and pharmacokinetics”, Adv. Drug Delivery Rev. 39(1-3):117-151 (1999); Taylor, “Improved passive oral drug delivery viaprodrugs”, Adv. Drug Delivery Rev., 19(2): 131-148 (1996); Valentino andBorchardt, “Prodrug strategies to enhance the intestinal absorption ofpeptides”, Drug Discovery Today 2(4): 148-155 (1997); Wiebe and Knaus,“Concepts for the design of anti-HIV nucleoside prodrugs for treatingcephalic HIV infection”, Adv. Drug Delivery Rev.: 39(1-3):63-80 (1999);Waller et al., “Prodrugs”, Br. J. Clin. Pharmac. 28: 497-507 (1989),content of all of which is herein incorporated by reference in itsentirety.

Pharmaceutical Compositions

For administration to a subject, the RAR agonists can be provided inpharmaceutically acceptable compositions. These pharmaceuticallyacceptable compositions comprise a therapeutically-effective amount ofone or more of the RAR agonists, formulated together with one or morepharmaceutically acceptable carriers (additives) and/or diluents. Asdescribed in detail below, the pharmaceutical compositions of thepresent invention can be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), gavages, lozenges, dragees, capsules, pills,tablets (e.g., those targeted for buccal, sublingual, and systemicabsorption), boluses, powders, granules, pastes for application to thetongue; (2) parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation; (3)topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin; (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually; (6) ocularly; (7) transdermally; (8)transmucosally; or (9) nasally. Additionally, compounds can be implantedinto a patient or injected using a drug delivery system. See, forexample, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236(1984); Lewis, ed. “Controlled Release of Pesticides andPharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No.3,773,919; and U.S. Pat. No. 35 3,270,960, content of all of which isherein incorporated by reference.

As used here, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar, (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water. (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

The phrase “effective-amount” as used herein, refers to an amount of anactive agent (e.g., an RARγ agonist) sufficient to produce the desiredchange or effect (e.g., priming mesenchymal stem cells toward myogenicdifferentiation in vitro).

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a RAR agonistwhich is effective for producing some desired therapeutic effect in atleast a sub-population of cells in an animal at a reasonablebenefit/risk ratio applicable to any medical treatment. For example, anamount of a RARγ agonist administered to a subject that is sufficient toproduce a statistically significant, measurable muscle repair orregeneration.

As used herein, the term “repair” refers to a process by which thedamages of a muscle tissue are alleviated or completely eliminated. Insome embodiments, at least one symptom of muscle tissue damage isalleviated by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, or at least 50%. Symptoms of muscle damage are described, forexample, in “Skeletal Muscle Damage and Repair” Tiidus, P. M., ed.,Human Kinetics (2008), content of which is herein incorporated byreference.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the composition at a desired site suchthat desired effect is produced. Routes of administration suitable forthe methods of the invention include both local and systemicadministration. Generally, local administration results in more RARagonist (or RAR agonist treated cells) being delivered to a specificlocation as compared to the entire body of the subject, whereas,systemic administration results in delivery of a RAR agonist (or RARagonist treated cells) to essentially the entire body of the subject.One method of local administration is by intramuscular injection.

In the context of administering a RAR agonist treated cell, the term“administering” also include transplantation of such a cell into asubject. As used herein, the term “transplantation” refers to theprocess of implanting or transferring at least one cell into a subject.The term “transplantation” includes, e.g., autotransplantation (removaland transfer of cell(s) from one location on a patient to the same oranother location on the same patient), allotransplantation(transplantation between members of the same species), andxenotransplantation (transplantations between members of differentspecies). Skilled artisan is well aware of methods for implanting ortransplantation of mesenchymal stem cells for muscle repair andregeneration, which are amenable to the present invention. See forexample, U.S. Pat. No. 7,592,174 and U.S. Pat. Pub. No. 2005/0249731,content of both of which is herein incorporated by reference.

Furthermore, the RAR agonist can be formulated in the form of ointments,creams powders, or other formulations suitable for topical formulations.Because the molecular weight of RAR agonists is generally less than 500daltons, these formulations can deliver the agonist from skin to deepermuscle tissue. Accordingly, such formulations may comprise one or moreagents that enhance penetration of active ingredient through skin. Fortopical applications, the RAR agonist can be included in wound dressingsand/or skin coating compositions.

A compound or composition described herein can be administered by anyappropriate route known in the art including, but not limited to, oralor parenteral routes, including intravenous, intramuscular,subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal,and topical (including buccal and sublingual) administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments of the aspects described herein, the compositionsare administered by intravenous infusion or injection.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments of the aspects describedherein, the subject is a mammal, e.g., a primate, e.g., a human. Theterms, “patient” and “subject” are used interchangeably herein. Theterms, “patient” and “subject” are used interchangeably herein. Asubject can be male or female.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofdisorders associated with autoimmune disease or inflammation. Inaddition, the methods and compositions described herein can be used totreat domesticated animals and/or pets.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a disorder characterized withmuscle damage or muscle atrophy/wasting.

A subject can be one who is not currently being treated with a RARagonist.

A subject can be one who has been previously diagnosed with a diseasethat is being treated with a therapeutic regime comprising a RARagonist, wherein the disease is not a disease characterized with muscledamage or muscle atrophy/wasting

Accordingly, in some embodiments, the treatment method comprisingadjusting the therapeutic regime of the subject such that at least onesymptom of muscle damage is reduced. Without limitation, a therapeuticregime can be adjusted by modulating the frequency of administration ofthe RAR and/or by altering the site or mode of administration.

In some embodiments of the aspects described herein, the method furthercomprising diagnosing a subject for muscle damage or muscleatrophy/wasting before treating the subject for muscle repair orregeneration.

In some embodiments of the aspects described herein, the method furthercomprising selecting a subject with muscle damage or muscleatrophy/wasting before treating the subject for muscle repair orregeneration.

Combination Therapy

In some embodiments of the aspects described herein, the RAR agonist isadministered to the subject along with a therapy selected from massage,ultrasound, hyperbaric oxygen delivery. In addition, and/oralternatively, the RAR agonist can be administrated to a subject incombination with a pharmaceutically active agent. Exemplarypharmaceutically active compound include, but are not limited to, thosefound in Harrison's Principles of Internal Medicine, 13^(th) Edition,Eds. T. R. Harrison et al. McGraw-Hill N.Y., NY; Physicians DeskReference, 50^(th) Edition, 1997, Oradell N.J., Medical Economics Co.;Pharmacological Basis of Therapeutics, 8^(th) Edition, Goodman andGilman, 1990; United States Pharmacopeia, The National Formulary, USPXII NF XVII, 1990; current edition of Goodman and Oilman's ThePharmacological Basis of Therapeutics; and current edition of The MerckIndex, the complete content of all of which are herein incorporated inits entirety.

In some embodiments of the aspects described herein, thepharmaceutically active agent include those agents known in the art fortreatment of inflammation or inflammation associated disorders, orinfections. Exemplary anti-inflammatory agents include, but are notlimited to, non-steroidal anti-inflammatory drugs (NSAIDs—such asaspirin, ibuprofen, or naproxen, coricosteroids (such as presnisone),anti-malarial medication (such as hydrochloroquine), methotrexrate,sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamise andmycophenolate.

In some embodiments of the aspects described herein, thepharmaceutically active agent is a growth factor. Exemplary growthfactors include, but are not limited to, fibroblast growth factors(FGF), FGF-1, FGF-2. FGF-4, thymosins, platelet-derived growth factors(PDGF), insulin binding growth factors (IGF), IGF-1, IGF-2, epidermalgrowth factor (EGF), transforming growth factor (TGF), TGF-.alpha.,TGF-.beta., cartilage inducing factors-A and -B, osteoid-inducingfactors, osteogenin, bone morphogenic proteins, and other bone growthfactors, collagen growth factors, heparin-binding growth factor-1 or -2,and their biologically active derivatives.

In some embodiments of the aspects described herein, thepharmaceutically active agent is an anti-interferon agent. Withoutlimitation, anti-interferon agents include anti-interferon antibodies orfragments or derivatives thereof. Exemplary anti-interferon antibodiesinclude, but are not limited to, those described in Ronnblom, L. &Elkon, K. B. Cytokines as therapeutic targets in SLE. Nat Rev Rheumatol6, 339-647; Yao, Y. et al. Neutralization ofinterferon-alpha/beta-inducible genes and downstream effect in a phase Itrial of an anti-interferon-alpha monoclonal antibody in systemic lupuserythematosus. Arthritis Rheum 60, 1785-96 (2009); and Zagury, D. et al.IFNalpha kinoid vaccine-induced neutralizing antibodies prevent clinicalmanifestations in a lupus flare murine model. Proc Natl Acad Sci USA106, 5294-9 (2009), those described in U.S. Pat. Nos. 4,902,618;5,055,289; 7,087,726; and 7,741,449, and those described in U.S. patentapplication Pub. Ser. No. 10/440,202; Ser. No. 11/342,020; and Ser. No.12/517,334, content of all of which is herein incorporated by reference.

In some embodiments of the aspects described herein, thepharmaceutically active agent is fenoterol or insulin-like growthfactor-1.

The RAR agonist and the pharmaceutically active agent can beadministrated to the subject in the same pharmaceutical composition orin different pharmaceutical compositions (at the same time or atdifferent times). When administered at different times, the RARγ agonistand the pharmaceutically active agent can be administered within 5minutes, 10 minutes, 20 minutes, 60 minutes, 2 hours, 3 hours, 4, hours,8 hours, 12 hours, 24 hours of administration of the other. When the RARagonist and the pharmaceutically active agent are administered indifferent pharmaceutical compositions, routes of administration can bedifferent.

Dosage

The amount of RAR agonist that can be combined with a carrier materialto produce a single dosage form will generally be that amount of the RARagonist that produces a therapeutic effect. Generally out of one hundredpercent, this amount will range from about 0.01% to 99% of RAR agonist,preferably from about 5% to about 70%, most preferably from 10% to about30%.

Toxicity and therapeutic efficacy can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compositions that exhibit large therapeutic indices, are preferred.

As used herein, the term ED denotes effective dose and is used inconnection with animal models. The term EC denotes effectiveconcentration and is used in connection with in vitro models.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

The therapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmamay be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay.

The dosage may be determined by a physician and adjusted, as necessary,to suit observed effects of the treatment. Generally, the compositionsare administered so that RARγ agonist is given at a dose from 1 μg/kg to150 mg/kg, 1 μg/kg to 100 mg/kg, 1 μg/kg to 50 mg/kg, 1 μg/kg to 20mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1 mg/kg, 100 μg/kg to 100 mg/kg,100 μg/kg to 50 mg/kg, 100 μg/kg to 20 mg/kg, 100 μg/kg to 10 mg/kg, 100μg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50mg/kg, or 10 mg/kg to 20 mg/kg. It is to be understood that ranges givenhere include all intermediate ranges, for example, the range 1 mg/kg to10 mg/kg includes 1 mg/kg to 2 mg/kg, 1 mg/kg to 3 mg/kg, 1 mg/kg to 4mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 6 mg/kg, 1 mg/kg to 7 mg/kg, 1mg/kg to 8 mg/kg, 1 mg/kg to 9 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 10mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 6 mg/kg to 10 mg/kg, 7mg/kg to 10 mg/kg, 8 mg/kg to 10 mg/kg, 9 mg/kg to 10 mg/kg, and thelike. It is to be further understood that the ranges intermediate to thegiven above are also within the scope of this invention, for example, inthe range 1 mg/kg to 10 mg/kg, dose ranges such as 2 mg/kg to 8 mg/kg, 3mg/kg to 7 mg/kg, 4 mg/kg to 6 mg/kg, and the like.

In some embodiments, the compositions are administered at a dosage sothat RAR agonist or a metabolite thereof has an in vivo concentration ofless than 500 nM, less than 400 nM, less than 300 nM, less than 250 nM,less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM,less than 25 nM, less than 20, nM, less than 10 nM, less than 5 nM, lessthan 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05, less than0.01, nM, less than 0.005 nM, less than 0.001 nM after 15 mins, 30 mins,1 hr, 1.5 hrs, 2 hrs, 2.5 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs,9 hrs, 10 hrs, 11 hrs, 12 hrs or more of time of administration.

With respect to duration and frequency of treatment, it is typical forskilled clinicians to monitor subjects in order to determine when thetreatment is providing therapeutic benefit, and to determine whether toincrease or decrease dosage, increase or decrease administrationfrequency, discontinue treatment, resume treatment or make otheralteration to treatment regimen. The dosing schedule can vary from oncea week to daily depending on a number of clinical factors, such as thesubject's sensitivity to the RAR agonist. The desired dose can beadministered everyday or every third, fourth, fifth, or sixth day. Thedesired dose can be administered at one time or divided into subdoses,e.g., 2-4 subdoses and administered over a period of time, e.g., atappropriate intervals through the day or other appropriate schedule.Such sub-doses can be administered as unit dosage forms. In someembodiments of the aspects described herein, administration is chronic,e.g., one or more doses daily over a period of weeks or months. Examplesof dosing schedules are administration daily, twice daily, three timesdaily or four or more times daily over a period of 1 week, 2 weeks, 3weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6months or more.

In one embodiment, administration of the RAR agonist to the subject inthe methods described herein is at one or more times. In one embodiment,one time of administration, or a plurality of times of theadministration, are within a time period of detectable increasedendogenous retinoid signaling that occurs naturally in vivo in responseto the damage (e.g., an incurred trauma or physiological change thatleads to muscle damage) to the muscle tissue (e.g., by day 4 or day 5after the incurrence of the damage). In one embodiment, administrationincludes a time that is at the beginning of the time period. In oneembodiment, administration includes several times that are within thetime period. In one embodiment, administration is at or includes a timethat is after a period of time that is greater than 3 days afterincurrence of the damage. In one embodiment, administration is at orincludes a time that is about 4 days after incurrence of the damage. Inone embodiment, administration is at or includes a time that is at about5 days after incurrence of the damage. In one embodiment, administrationis at or includes a time that is at about 6 days after incurrence of thedamage. In one embodiment, administration is at or includes a time thatis at about 7, or about 8 days after incurrence of the damage.Combinations of these times of administration are also envisioned. Forexample, administration at about day 4, and then one or more of days 5,6, 7, and 8. In one embodiment, administration is at about day 5 and day7. Continued administration over an extended period of time (e.g., untilsatisfactory recovery is obtained) can also be performed.

In one embodiment, the administration overlaps with or encompasses thetime period of increased endogenous retinoid signaling (e.g.,administration begins prior to the increased endogenous retinoidsignaling, and extends into and/or throughout this period). In oneembodiment, the administration is withheld until the increasedendogenous retinoid signaling in response to the damage to the muscletissue (e.g. administration begins following day 3, or at day 4, 5, 6,7, 8, or 9 or later). In one embodiment, administration begins followingday 3 (e.g., day 4 or 5), and is continued through to at least day 9(e.g., until day 11). In one embodiment, administration is every day orevery other day during the recited time periods of administration. Inone embodiment, administration is at least on day 5, 7, and 9.

Pre-Treated Stem Cells

In another aspect, the invention provides a population of stem cells,wherein the population is produced by contacting at least one cell stemcell with an effective amount of a RAR agonist. As used herein, the term“population of stem cells” means one or more stem cells. Such stem cellscan be isolated, (e.g., for contacting in vitro, or ex vivo). Suchcontacting enables the cells to regenerate muscle tissue and also tosupport myogenic differentiation of other cells (e.g., cells in a hostinto which the pre-treated cells are administered). In one embodiment,the contact is with RARγ agonist, to isolated mesenchymal stem cells, toenable this ability. In one embodiment, pre-treatment is in vitro and isfor at least about 3 days. Although shorter periods of pre-treatment(e.g., 2.5 days, 2 days, 1.5 days, 1 day, 12 hours) may also producesimilar abilities. Such pre-treated cells which are thus induced, arereferred to herein as “pre-treated”. All pre-treated cells describedherein are encompassed by the present invention.

The term “contacting” or “contact” as used herein in connection withcontacting a stem cell includes subjecting the stem cell to anappropriate culture media which comprises a RAR agonist. The stem cellcan be contacted with an RAR agonist in a cell culture, e.g., in vitroor ex vivo. As used herein, the term “ex vivo” refers to cells which areremoved from a living organism and cultured outside the organism (e.g.,in a test tube). Ex vivo cells can then be administered uponmodification to the donor.

In some embodiments of the aspects of the invention, the stem cell is ahuman stem cell. In some embodiments, the stem cell is a pluripotent ormultipotent stem cell. In some embodiments, the stem cell is an inducedpluripotent stem (iPS) cell, or a stably reprogrammed cell which is anintermediate pluripotent stem cell and can be further reprogrammed intoan iPS cell, e.g., partial induced pluripotent stem cells (also referredto as “piPS cells”). In some embodiments, the pluripotent stem cell,iPSC or piPSC is a genetically modified pluripotent stem cell. In someembodiments, the stem cell is a mesenchymal stem cell.

Generally, a stem cell for use in the methods, assays, systems, kits andto generate scorecards can be obtained or derived from any availablesource. Accordingly, a stem cell can be obtained or derived from avertebrate or invertebrate. In some embodiments of this and otheraspects of the invention, the stem cell is a mammalian stem cell.

In some embodiments of this and other aspects of the invention, the stemcell is a primate or a rodent stem cell.

In some embodiments of the aspects of the invention, the stem cell isselected from the group consisting of chimpanzee, cynomologous monkey,spider monkey, macaques (e.g. Rhesus monkey), mouse, rat, woodchuck,ferret, rabbit, hamster, cow, horse, pig, deer, bison, buffalo, feline(e.g., domestic cat), canine (e.g. dog, fox and wolf), avian (e.g.chicken, emu, and ostrich), and fish (e.g., trout, catfish and salmon)stem cell.

As used herein, the term “differentiation” as used herein refers to thecellular development of a cell from a primitive stage towards a moremature (i.e. less primitive) cell.

As used here in, the term “induced pluripotent stem cell” or “iPSC” or“iPS cell” refers to a cell derived from a complete reversion orreprogramming of the differentiation state of a differentiated cell(e.g. a somatic cell). As used herein, an iPSC is fully reprogrammed andis a cell which has undergone complete epigenetic reprogramming.

The term “reprogramming” as used herein refers to a process that altersor reverses the differentiation state of a differentiated cell (e.g. asomatic cell). Stated another way, reprogramming refers to a process ofdriving the differentiation of a cell backwards to a moreundifferentiated or more primitive type of cell. Complete reprogramminginvolves complete reversal of at least some of the heritable patterns ofnucleic acid modification (e.g., methylation), chromatin condensation,epigenetic changes, genomic imprinting, etc., that occur during cellulardifferentiation as a zygote develops into an adult. Reprogramming isdistinct from simply maintaining the existing undifferentiated state ofa cell that is already pluripotent or maintaining the existing less thanfully differentiated state of a cell that is already a multipotent cell(e.g., a hematopoietic stem cell). Reprogramming is also distinct frompromoting the self-renewal or proliferation of cells that are alreadypluripotent or multipotent, although the compositions and methods of theinvention may also be of use for such purposes.

The term “stable reprogrammed cell” as used herein refers to a cellwhich is produced from the partial or incomplete reprogramming of adifferentiated cell (e.g. a somatic cell). A stable reprogrammed cell isused interchangeably herein with “piPSC”. A stable reprogrammed cell hasnot undergone complete reprogramming and thus has not had globalremodeling of the epigenome of the cell. A stable reprogrammed cell is apluripotent stem cell and can be further reprogrammed to an iPSC, asthat term is defined herein, or alternatively can be differentiatedalong different lineages. In some embodiments, a partially reprogrammedcell expresses markers from all three embryonic germ layers (i.e. allthree layers of endoderm, mesoderm or ectoderm layers). Markers ofendoderm cells include, Gata4, Fox A2, PDX1, Nodal, Sox7 and Sox17.Markers of mesoderm cells include, Brachycury, GSC, LEF1, Mox1 and Tie1.Markers of ectoderm cells include cripto1, EN1, GFAP. Islet 1, LIM1 andNestin. In some embodiments, a partially reprogrammed cell is anundifferentiated cell.

The term “remodeling of the epigenome” refers to chemical modificationsof the genome which do not change the genomic sequence or a gene'ssequence of base pairs in the cell, but alter the expression.

The term “global remodeling of the epigenome” refers to where chemicalmodifications of the genome have occurred where there is no memory ofprior gene expression from the differentiated cell from which thereprogrammed cell or iPSC was derived.

The term “incomplete remodeling of the epigenome” refers to wherechemical modifications of the genome have occurred where there is memoryof prior gene expression from the differentiated cell from which thestable reprogrammed cell or piPSC was derived.

The term “epigenetic reprogramming” as used herein refers to thealteration of the pattern of gene expression in a cell via chemicalmodifications that do not change the genomic sequence or a gene'ssequence of base pairs in the cell.

The term “epigenetic” as used herein refers to “upon the genome”.Chemical modifications of DNA that do not alter the gene's sequence, butimpact gene expression and may also be inherited. Epigenetic, alsocalled posttranslational modifications or “PTM” to DNA are important,for example, in imprinting and cellular reprogramming. Thesemodifications include, for example, DNA methylation, ubiquitination,phosphorylation, glycosylation, sumoylation, acetylation,S-nitrosylation or nitrosylation, citrullination or deimination,neddylation, OClcNAc, ADP-ribosylation, hydroxylation, fattenylation,ufmylation, prenylation, myristoylation, S-palmitoylation, tyrosinesulfation, formylation, and carboxylation.

The term “pluripotent” as used herein refers to a cell with thecapacity, under different conditions, to differentiate to cell typescharacteristic of all three germ cell layers (endoderm, mesoderm andectoderm). Pluripotent cells are characterized primarily by theirability to differentiate to all three germ layers, using, for example, anude mouse teratoma formation assay. Pluripotency is also evidenced bythe expression of embryonic stem (ES) cell markers, although thepreferred test for pluripotency is the demonstration of the capacity todifferentiate into cells of each of the three germ layers. In someembodiments, a pluripotent cell is an undifferentiated cell.

The term “pluripotency” or a “pluripotent state” as used herein refersto a cell with the ability to differentiate into all three embryonicgerm layers: endoderm (gut tissue), mesoderm (including blood, muscle,and vessels), and ectoderm (such as skin and nerve), and typically hasthe potential to divide in vitro for a long period of time, e.g.,greater than one year or more than 30 passages.

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that is able to differentiate into some but not all ofthe cells derived from all three germ layers. Thus, a multipotent cellis a partially differentiated cell. Multipotent cells are well known inthe art, and examples of multipotent cells include adult stem cells,such as for example, hematopoietic stem cells and neural stem cells.Multipotent means a stem cell may form many types of cells in a givenlineage, but not cells of other lineages. For example, a multipotentblood stem cell can form the many different types of blood cells (red,white, platelets, etc. . . . ), but it cannot form neurons.

The term “multipotency” refers to a cell with the degree ofdevelopmental versatility that is less than totipotent and pluripotent.

The term “totipotency” refers to a cell with the degree ofdifferentiation describing a capacity to make all of the cells in theadult body as well as the extra-embryonic tissues including theplacenta. The fertilized egg (zygote) is totipotent as are the earlycleaved cells (blastomeres).

The term “differentiated cell” is meant any primary cell that is not, inits native form, pluripotent as that term is defined herein. The term a“differentiated cell” also encompasses cells that are partiallydifferentiated, such as multipotent cells, or cells that are stablenon-pluripotent partially reprogrammed cells. It should be noted thatplacing many primary cells in culture can lead to some loss of fullydifferentiated characteristics. Thus, simply culturing such cells areincluded in the term differentiated cells and does not render thesecells non-differentiated cells (e.g. undifferentiated cells) orpluripotent cells. The transition of a differentiated cell topluripotency requires a reprogramming stimulus beyond the stimuli thatlead to partial loss of differentiated character in culture.Reprogrammed cells also have the characteristic of the capacity ofextended passaging without loss of growth potential, relative to primarycell parents, which generally have capacity for only a limited number ofdivisions in culture. In some embodiments, the term “differentiatedcell” also refers to a cell of a more specialized cell type derived froma cell of a less specialized cell type (e.g., from an undifferentiatedcell or a reprogrammed cell) where the cell has undergone a cellulardifferentiation process.

As used herein, the term “somatic cell” refers to any cell other than agerm cell, a cell present in or obtained from a pre-implantation embryo,or a cell resulting from proliferation of such a cell in vitro. Statedanother way, a somatic cell refers to any cells forming the body of anorganism, as opposed to germline cells. In mammals, germline cells (alsoknown as “gametes”) are the spermatozoa and ova which fuse duringfertilization to produce a cell called a zygote, from which the entiremammalian embryo develops. Every other cell type in the mammalianbody-apart from the sperm and ova, the cells from which they are made(gametocytes) and undifferentiated stem cells—is a somatic cell:internal organs, skin, bones, blood, and connective tissue are all madeup of somatic cells. In some embodiments the somatic cell is a“non-embryonic somatic cell”, by which is meant a somatic cell that isnot present in or obtained from an embryo and does not result fromproliferation of such a cell in vitro. In some embodiments the somaticcell is an “adult somatic cell”, by which is meant a cell that ispresent in or obtained from an organism other than an embryo or a fetusor results from proliferation of such a cell in vitro. Unless otherwiseindicated the methods for reprogramming a differentiated cell can beperformed both in vivo and in vitro (where in vivo is practiced when andifferentiated cell is present within a subject, and where in vitro ispracticed using isolated differentiated cell maintained in culture). Insome embodiments, where a differentiated cell or population ofdifferentiated cells are cultured in vitro, the differentiated cell canbe cultured in an organotypic slice culture, such as described in, e.g.,Meneghel-Rozzo et al., (2004), Cell Tissue Res, 316(3); 295-303, whichis incorporated herein in its entirety by reference.

As used herein, the term “adult cell” refers to a cell found throughoutthe body after embryonic development.

In the context of cell ontogeny, the term “differentiate”, or“differentiating” is a relative term meaning a “differentiated cell” isa cell that has progressed further down the developmental pathway thanits precursor cell. Thus in some embodiments, a reprogrammed cell asthis term is defined herein, can differentiate to lineage-restrictedprecursor cells (such as a mesodermal stem cell), which in turn candifferentiate into other types of precursor cells further down thepathway (such as an tissue specific precursor, for example, acardiomyocyte precursor), and then to an end-stage differentiated cell,which plays a characteristic role in a certain tissue type, and may ormay not retain the capacity to proliferate further.

The term “embryonic stem cell” is used to refer to the pluripotent stemcells of the inner cell mass of the embryonic blastocyst (see U.S. Pat.Nos. 5,843,780, 6,200,806, which are incorporated herein by reference).Such cells can similarly be obtained from the inner cell mass ofblastocysts derived from somatic cell nuclear transfer (see, forexample, U.S. Pat. Nos. 5,945,577, 5,994,619, 6,235,970, which areincorporated herein by reference). The distinguishing characteristics ofan embryonic stem cell define an embryonic stem cell phenotype.Accordingly, a cell has the phenotype of an embryonic stem cell if itpossesses one or more of the unique characteristics of an embryonic stemcell such that that cell can be distinguished from other cells.Exemplary distinguishing embryonic stem cell characteristics include,without limitation, gene expression profile, proliferative capacity,differentiation capacity, karyotype, responsiveness to particularculture conditions, and the like.

The term “phenotype” refers to one or a number of total biologicalcharacteristics that define the cell or organism under a particular setof environmental conditions and factors, regardless of the actualgenotype.

The term “isolated cell” as used herein refers to a cell that has beenremoved from an organism in which it was originally found or adescendant of such a cell. Optionally the cell has been cultured invitro, e.g., in the presence of other cells. Optionally the cell islater introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells as used herein refers to a population of cells that has beenremoved and separated from a mixed or heterogeneous population of cells.In some embodiments, an isolated population is a substantially purepopulation of cells as compared to the heterogeneous population fromwhich the cells were isolated or enriched from.

The term “substantially pure”, with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,preferably at least about 85%, more preferably at least about 90%, andmost preferably at least about 95% pure, with respect to the cellsmaking up a total cell population. Recast, the terms “substantiallypure” or “essentially purified”, with regard to a population of cells,refers to a population of cells that contain fewer than about 20%, morepreferably fewer than about 15%, 10%, 8%, 7%, most preferably fewer thanabout 5%, 4%, 3%, 2%, 1%, or less than 1%, of cells that are not theindicated cells or their progeny as defined by the terms herein.

The terms “enriching” or “enriched” are used interchangeably herein andmean that the yield (fraction) of cells of one type is increased by atleast 10% over the fraction of cells of that type in the startingculture or preparation.

The terms “renewal” or “self-renewal” or “proliferation” are usedinterchangeably herein, and refers to a process of a cell making morecopies of itself (e.g. duplication) of the cell. In some embodiments,reprogrammed cells are capable of renewal of themselves by dividing intothe same undifferentiated cells (e.g. pluripotent or non-specializedcell type) over long periods, and/or many months to years. In someinstances, proliferation refers to the expansion of reprogrammed cellsby the repeated division of single cells into two identical daughtercells.

The term “cell culture medium” (also referred to herein as a “culturemedium” or “medium”) as referred to herein is a medium for culturingcells containing nutrients that maintain cell viability and supportproliferation. The cell culture medium may contain any of the followingin an appropriate combination: salt(s), buffer(s), amino acids, glucoseor other sugar(s), antibiotics, serum or serum replacement, and othercomponents such as peptide growth factors, etc. Cell culture mediaordinarily used for particular cell types are known to those skilled inthe art.

The term “lineages” as used herein describes a cell with a commonancestry or cells with a common developmental fate. By way of an exampleonly, a cell that is of endoderm origin or is “endodermal linage” thismeans the cell was derived from an endodermal cell and can differentiatealong the endodermal lineage restricted pathways, such as one or moredevelopmental lineage pathways which give rise to definitive endodermcells, which in turn can differentiate into liver cells, thymus,pancreas, lung and intestine.

A stem cell (e.g., an isolated stem cell such as an embryonic stem cellisolated from a subject) can be contacted with a RAR agonist for anyamount of time. For example, a stem cell can be contacted with a RARagonist for 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1week or more. In one non-limiting example, RAR agonists (dissolved inethanol or DMSO) are added to MSC cultures at the final concentrationbetween 10 nM and 3 μM, and the volume of RAR agonist solution added tothe culture is 0.1%.

Without wishing to be bound by a theory, the contacting a stem cell witha RAR agonist induces or stimulates lineage-directed differentiation ofthe stem cell into particular lineage. For example, the contacting astem cell with a RAR agonist can induce or stimulate differentiationinto a lineage selected from the group consisting of mesoderm, endoderm,ectoderm, neuronal, mesenchymal, and hematopoietic lineage.

In some embodiments of this and other aspects of the invention, theinduced/stimulated lineage is a mesenchymal lineage.

In some embodiments, the induced mesenchymal lineage is selected fromthe group consisting of myogenic, osteogenic, chodrogenic, tendonogenic,ligamentogenic, maroow stromagenic, adipogenic, and dermogenic.

As used herein, a “mesenchymal stem cell” is a mesenchymal cell havingthe ability to autoreproduce and to differentiate into one or moremesenchymal cells. Like a mesodermal cell, a mesenchymal stem cell ispluripotent, capable of differentiating into osteoblasts, cartilagecells, myoblasts, fat cells, stroma cells, tendon cells, and the like.While autoreproducing and pluripotent mesodermal cells lose theseabilities in the process of development, mesenchymal stem cells areknown to persist for a long time in the adult body after it has passedthrough development.

As used herein, “mesenchymal cells” are ostoblasts, cartilage cells,myoblasts, fat cells, stroma cells, tendon cells and other cells thatform mesenchymal tissue, and mesenchymal stem cells which maydifferentiate into these. Mesenchymal cells occurring duringembryogenesis, mesenchymal cells in individual animals, and mesenchymalcells generated by differentiation from pluripotent stem cells in vitroor in vivo are all included in the term “mesenchymal cells.”

The stem cells can come or obtained from any source available to thepractitioner.

Mesenchymal stem cells can be obtained by a number of methods well knownin the art. See for example, U.S. Pat. Nos. 5,486,358; 6,387,367;and7,592,174, and U.S. Pat App. Pub. No. 2003/0211602, content of all ofwhich is herein incorporated by reference. Mesenchymal cells can includeautologous mesenchymal stem cells, i.e., a cell or cells taken from asubject who is in need of treatment (i.e., the donor and recipient arethe same individual). Autologus mesenchymal stem cells have theadvantage of avoiding any immunologically-based rejection of the cells.Alternatively, the cells can be heterologous, e.g., taken from a donor.The second subject can be of the same or different species. Typically,when the cells come from a donor, they will be from a donor who issufficiently immunologically compatible with the recipient, i.e., willnot be subject to transplant rejection, to lessen or remove the need forimmunosuppression. In some embodiments, the cells are taken from axenogeneic source, i.e., a non-human mammal that has been geneticallyengineered to be sufficiently immunologically compatible with therecipient, or the recipient's species. Methods for determiningimmunological compatibility are known in the art, and include tissuetyping to assess donor-recipient compatibility for HLA and ABOdeterminants. See, e.g., Transplantation Immunology, Bach andAuchincloss, Eds. (Wiley. John & Sons, Incorporated 1994).

In some embodiments, the mesenchymal stem cell is derived from ade-differentiated somatic cell (a reprogrammed cell). For example, asomatic cell de-differentiated to a pluripotent stem cell, for exampleby direct reprogramming of a cell of endodermal origin. Without wishingto be bound by theory, a de-differentiated cell has a morphology thatresembles a more primitive cell type from which it was derived, e.g.,mesenchymal morphology.

In some embodiments, the mesenchymal stem cell is a re-differentiatedmesenchymal stem cell. As used herein, the term “re-differentiatedmesenchymal stem cell” refers to a mesenchymal stem cell that isdifferentiated from a de-differentiated mesenchymal stem cell.

In some embodiments, the mesenchymal stem cells are in a stabilizedstate, e.g., the cells were taken from a subject and treated in such amanner as to allow them to be stored for some period of time. Forexample, the cells can be frozen, e.g., using methods known in the artfor freezing primary cells, such that the cells are viable when thawed.For example, methods known in the art to freeze and thaw embryos togenerate live mammals can be adapted for use in the present methods.Such methods may include the use of liquid nitrogen, e.g., with one ormore cryoprotectants, e.g., agents that prevent freeze-thaw damage tothe cell.

The population of mesenchymal stem cells obtained from a subject ordonor can be substantially pure. The purity of the population can bedetermined, and manipulated, using methods known in the art. Forexample, methods using fluorescence activated cell sorting can be used.

Without wishing to be bound by a theory, any suitable cell culture mediacan be used for in vitro or ex vivo methods of the invention. Forexample, MSCs can be maintained in α-MEM medium containing 10% fetalbovie serum.

In some embodiments of this and other aspects of the invention,mesenchymal stem cell is a bone marrow-derived mesenchymal stem cell(BMSC).

In some embodiments of this and other aspects of the invention,mesenchymal stem cell is murine marrow derived mesenchymal stem cell.

In some embodiments of this and other aspects of the invention,mesenchymal stem cell is a human mesenchymal stem cell (hMSC).

The inventors have also discovered that RAR agonist treated stem cellscan be used for repairing or regenerating muscle. Accordingly, inanother aspect, the invention provides a method for muscle repair orregeneration in a subject, the method comprising administering apopulation of stem cells to a subject, wherein at least one cell in thepopulation has been contacted with a RAR agonist.

In some embodiments, the method comprises steps of: (i) contacting atleast one stem cell with a RAR agonist; and (ii) administering said stemcells to a subject, which subject has a damaged muscle tissue.

After contacting the at least 1 stem cell with the RAR agonist for theneeded time, treated stem cell can be administered right away or storedfor a period of time before administration to a subject. Withoutlimitation, the period of time can range from minutes to days.

The number of RAR agonist treated stem cells to be administered to asubject can range from a single cell to over 10⁶.

Another aspect of the invention relates to a method for muscle repair orregeneration in a subject, the method comprising administering apopulation of stem cells to a subject, wherein a portion of the stemcells are pre-treated with a RARγ agonist, as described herein. In oneembodiment, the portion of pre-treated stem cells is about 50% (a 1:1ratio). The use of populations of pre-treated stem cells with higherratios (e.g., 2:1, 3:1, 4:1, 5:1, pre-treated stem cell:untreated stemcell) and also with lower ratios (1:2, 1:3, 1:4, 1:5, pre-treated stemcell:untreated stem cell) is also seen as useful in the method. Forexample, the portion of pre-treated stem cells can be about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, or 45%. The portion of pre-treated stemcells can also be about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

The method of muscle repair or regeneration comprises administering thecomposition comprising the pre-treated and non-treated stem cells, to asubject at a site of muscle injury, to thereby repair or regenerate themuscle at the site. In one embodiment, the injury is a compound tissueinjury. A compound tissue injury is a site of multiple tissue damage,and can include one or more of muscle, bone, tendon, ligament, and fattissue injury. Various combinations are envisioned. In one embodiment,the compound tissue injury includes muscle and bone. In one embodiment,the compound tissue injury include muscle and at least one additionaltissue (e.g., ligaments, tendons, cartilage, bone, skin, fat and bloodvessels). In one embodiment, the compound tissue injury include muscleand at least two additional tissues (e.g., two or more of ligaments,tendons, cartilage, bone, skin, fat and blood vessels). In oneembodiment, the additional tissue injury includes bone. In oneembodiment, the compound tissue injury include muscle and at least threeadditional tissues (e.g., three or more of ligaments, tendons,cartilage, bone, skin, fat and blood vessels). In one embodiment, theadditional tissue injury includes bone. In one embodiment, the compoundtissue injury include muscle and at least four additional tissues. Inone embodiment, the additional tissue injury includes bone. Additionaltissue types other than those described herein may also be included inthe injury, in combination with the muscle injury.

As discussed above, the inventors have discovered that progenitor stemcells can be induced to undergo muscle differentiation by acute orchronic exposure to retinoic acid receptor gamma (RARγ) agonists.Accordingly, in one aspect, the invention provides a method of inducingor stimulating lineage-directed differentiation of a stem cell into asingle particular lineage, the method comprising contacting a stem cellwith a retinoic acid receptor agonist.

In some embodiments, of this and other aspects of the invention, theparticular lineage is selected from the group consisting of mesoderm,endoderm, ectoderm, neuronal, hematopoietic lineages, and anycombinations thereof.

In some embodiments of this and other aspects of the invention, theparticular lineage is a mesenchymal lineage.

In some embodiments of this and other aspects of the invention, themesenchymal lineage is selected from the group consisting of myogenic,osteogenic, chodrogenic, tendonogenic, ligamentogenic, maroowstromagenic, adipogenic, and dermogenic.

Kits

In another aspect, the invention provides a kit for muscle repair orregeneration. In some embodiments, the kit comprises a RAR agonist. Insome embodiments, the kit further comprises a population of stem cells.The RAR agonist can be pre-formulated into a pharmaceutical formulationfor administration or ingredients for formulating into a pharmaceuticalformulation can be provided in the kit.

In some embodiments, the kit comprises a RAR agonist, wherein theagonist is formulated for topical application.

In some embodiments, the kit comprises a population of stem cells,wherein at least one cell in the population has been pretreated bycontacting the cells with a RAR agonist.

In addition to the above mentioned components, the kit can includeinformational material. The informational material can be descriptive,instructional, marketing or other material that relates to the methodsdescribed herein and/or the use of the compound for the methodsdescribed herein. For example, the informational material describesmethods for administering the formulation to a subject. The kit can alsoinclude a delivery device.

In one embodiment, the informational material can include instructionsto administer the formulation in a suitable manner, e.g., in a suitabledose, dosage form, or mode of administration (e.g., a dose, dosage form,or mode of administration described herein). In another embodiment, theinformational material can include instructions for identifying asuitable subject, e.g., a human, e.g., an adult human. The informationalmaterial of the kits is not limited in its form. In many cases, theinformational material, e.g., instructions, is provided in printedmatter, e.g., a printed text, drawing, and/or photograph, e.g., a labelor printed sheet. However, the informational material can also beprovided in other formats, such as Braille, computer readable material,video recording, or audio recording. In another embodiment, theinformational material of the kit is a link or contact information.e.g., a physical address, email address, hyperlink, website, ortelephone number, where a user of the kit can obtain substantiveinformation about the formulation and/or its use in the methodsdescribed herein. Of course, the informational material can also beprovided in any combination of formats.

In some embodiments the individual components of the formulation can beprovided in one container. Alternatively, it can be desirable to providethe components of the formulation separately in two or more containers,e.g., one container for an oligonucleotide preparation, and at leastanother for a carrier compound. The different components can becombined, e.g., according to instructions provided with the kit. Thecomponents can be combined according to a method described herein, e.g.,to prepare and administer a pharmaceutical composition.

In addition to the formulation, the composition of the kit can includeother ingredients, such as a solvent or buffer, a stabilizer or apreservative, and/or a second agent for treating a condition or disorderdescribed herein. Alternatively, the other ingredients can be includedin the kit, but in different compositions or containers than theformulation. In such embodiments, the kit can include instructions foradmixing the formulation and the other ingredients, or for using theoligonucleotide together with the other ingredients.

The RAR agonists can be provided in any form, e.g., liquid, dried orlyophilized form. It is preferred that the formulation be substantiallypure and/or sterile. When the formulation is provided in a liquidsolution, the liquid solution preferably is an aqueous solution, with asterile aqueous solution being preferred. When the formulation isprovided as a dried form, reconstitution generally is by the addition ofa suitable solvent. The solvent, e.g., sterile water or buffer, canoptionally be provided in the kit.

In some embodiments, the kit contains separate containers, dividers orcompartments for the formulation and informational material. Forexample, the formulation can be contained in a bottle, vial, or syringe,and the informational material can be contained in a plastic sleeve orpacket. In other embodiments, the separate elements of the kit arecontained within a single, undivided container. For example, theformulation is contained in a bottle, vial or syringe that has attachedthereto the informational material in the form of a label.

In some embodiments, the kit includes a plurality, e.g., a pack, ofindividual containers, each containing one or more unit dosage forms ofthe formulation. For example, the kit includes a plurality of syringes,ampules, foil packets, or blister packs, each containing a single unitdose of the formulation. The containers of the kits can be air tightand/or waterproof.

Definitions

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments of the aspects described herein, andare not intended to limit the claimed invention, because the scope ofthe invention is limited only by the claims. Further, unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “elevated,” means an increase by a statically significantamount: for the avoidance of any doubt, the term “elevated” means anincrease of at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 1-fold, atleast 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, or at least 10-fold or greater as compared to a referencelevel.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) above or below a reference level. The term refers to statisticalevidence that there is a difference. It is defined as the probability ofmaking a decision to reject the null hypothesis when the null hypothesisis actually true. The decision is often made using the p-value.

As used herein, the term “ex vivo” refers to cells which are removedfrom a living organism and cultured outside the organism (e.g., in atest tube).

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to described the present invention,in connection with percentages means±1%.

In one respect, the present invention relates to the herein describedcompositions, methods, and respective component(s) thereof, as essentialto the invention, yet open to the inclusion of unspecified elements,essential or not (“comprising). In some embodiments, other elements tobe included in the description of the composition, method or respectivecomponent thereof are limited to those that do not materially affect thebasic and novel characteristic(s) of the invention (“consistingessentially of”). This applies equally to steps within a describedmethod as well as compositions and components therein. In otherembodiments, the inventions, compositions, methods, and respectivecomponents thereof, described herein are intended to be exclusive of anyelement not deemed an essential element to the component, composition ormethod (“consisting of”).

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any one of the various embodiments hereindescribed and illustrated may be further modified to incorporatefeatures shown in any of the other embodiments disclosed herein.

All patents, patent applications, and publications identified areexpressly incorporated herein by reference for the purpose of describingand disclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

The present invention may be as defined in any one of the followingnumbered paragraphs.

-   1. A method of muscle repair or regeneration in a subject,    comprising administering a therapeutically effective amount of a    retinoic acid receptor gamma (RARγ) agonist to a subject with    damaged muscle tissue, to thereby repair or regenerate the damaged    muscle tissue.-   2. The method of paragraph 1, wherein said administration is local    or systemic.-   3. The method of any one of paragraphs 1-2, wherein administration    is begun during a time period of increased endogenous retinoid    signaling in the subject resulting from incurrence of the damaged    muscle tissue.-   4. The method of any one of paragraphs 1-3, wherein administration    is begun later than 3 days after incurrence of the damaged muscle    tissue by the subject.-   5. The method of paragraph 4, wherein administration is begun at    about 4 days after incurrence of the damaged muscle tissue by the    subject.-   6. The method of paragraph 4, wherein administration is begun at    about 5 days after incurrence of the damaged muscle tissue by the    subject.-   7. The method of paragraph 4, wherein administration is begun at    about 6 days after incurrence of the damaged muscle tissue by the    subject.-   8. The method of paragraph 4, wherein administration is begun at    about 7 days after incurrence of the damaged muscle tissue by the    subject.-   9. The method of paragraph 4, wherein administration is begun at    about 5 days, and is continued through to at least day 7 after    incurrence of the damaged muscle tissue by the subject.-   10. The method of paragraph 9, wherein administration is continued    through to at least day 9 after incurrence of the damaged muscle    tissue by the subject.-   11. The method of any one of paragraphs 1-10, wherein the RARγ    agonist is selected from the group consisting of CD-271    (6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic    acid); CD-394, CD-437    (6-(4-Hydroxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic    acid); CD-1530    (4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoic    acid); CD-2247; palovarotene    (4-[(1E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]-benzoic    acid); BMS-270394    (3-Fluoro-4-[(R)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic    acid); BMS-189961    (3-Fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic    acid); CH-55    (4-[(E)-3-(3,5-Di-tert-butyl-phenyl)-3-oxo-propenyl]-benzoic acid);    6-[3-(adamantan-1-yl)-4-(prop-2-ynyloxy)phenyl]naphthalene-2-carboxylic    acid;    5-[(E)-3-oxo-3-(5,5,8,8-tetrahydronaphthalene-2-yl)propenyl]thiophene-2-carboxylic    acid; and enantiomers, derivatives, prodrugs, and pharmaceutically    acceptable salts thereof.-   12. The method of any one of paragraphs 1-11, wherein the damaged    muscle tissue is the result of a physical injury or accident,    disease, infection, over-use, loss of blood circulation, or muscle    atrophy or wasting.-   13. The method of any one of paragraphs 1-11, wherein the damaged    muscle tissue is dystrophic muscle or an ageing muscle.-   14. The method of any one of paragraphs 1-11, wherein the damaged    muscle tissue is the result of muscle atrophy/wasting.-   15. The method of any one of paragraphs 1-14, wherein the subject is    a mammal.-   16. The method of any one of paragraphs 1-15, wherein the subject is    a mouse.-   17. The method of any one of paragraphs 1-15, wherein the subject is    human.-   18. The method of any one of paragraphs 1-17, further comprising    administering an anti-inflammatory agent to the subject.-   19. A method of muscle repair or regeneration in a subject,    comprising administering pluripotent or multipotent stem cells that    have been pre-treated with a RARγ agonist, to a subject at a site of    muscle injury, to thereby repair or regenerate muscle at the site.-   20. The method of paragraph 19, wherein the muscle injury is a    compound tissue injury.-   21. The method of paragraph 20, wherein the compound tissue injury    comprises an injury to muscle and bone.-   22. The method of any one of paragraphs 19-21, wherein the    pre-treated stem cells are administered in combination with    non-treated stem cells.-   23. The method of paragraph 22, wherein the pre-treated stem cells    and non-treated stem cells are administered at a ratio of 1:1.-   24. The method of any one of paragraphs 19-23, wherein the    pre-treated stem cells have been pre-treated with the RARγ agonist    for a period of about 3 days.-   25. The method of paragraph 19, wherein the pluripotent stem cells    are induced pluripotent stem cells.-   26. The method of any one of paragraphs 19-25, wherein the    pluripotent stem cells are mesenchymal stem cells.-   27. The method of any one of paragraphs 19-26, wherein administering    is local.-   28. The method of any one of paragraphs 19-27, wherein administering    is by transplantation of the cells into the subject.-   29. The method of any one of paragraphs 19-28, wherein the    pluripotent stem cells are autologous or heterologous to the    subject.-   30. The method of any one of paragraphs 19-29, wherein the    pluripotent stem cells are mammalian.-   31. The method of any one of paragraphs 19-30, wherein the    pluripotent stem cells are rodent.-   32. The method of any one of paragraphs 19-30, wherein the    pluripotent stem cells are human.-   33. The method of any one of paragraphs 19-32, further comprising    administering an anti-inflammatory agent to the subject.-   34. The method of any one of paragraphs 19-33, wherein the RARγ    agonist is selected from the group consisting of CD-271    (6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic    acid); CD-394, CD-437    (6-(4-Hydroxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic    acid); CD-1530    (4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoic    acid); CD-2247; palovarotene    (4-[(1E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]-benzoic    acid); BMS-270394    (3-Fluoro-4-[(R)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic    acid); BMS-189961    (3-Fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic    acid); CH-55    (4-[(E)-3-(3,5-Di-tert-butyl-phenyl)-3-oxo-propenyl]-benzoic acid);    6-[3-(adamantan-1-yl)-4-(prop-2-ynyloxy)phenyl]naphthalene-2-carboxylic    acid;    5-[(E)-3-oxo-3-(5,5,8,8-tetrahydronaphthalene-2-yl)propenyl]thiophene-2-carboxylic    acid; and enantiomers, derivatives, prodrugs, and pharmaceutically    acceptable salts thereof.-   35. The method of any one of claims 19-34 wherein the injured muscle    results from physical injury or accident, disease, infection,    over-use, loss of blood circulation, or muscle atrophy or wasting.-   36. The method of any one of paragraphs 19-34, wherein the injured    muscle tissue is dystrophic muscle or an ageing muscle.-   37. The method of any one of paragraphs 19-34, wherein the injured    muscle is the result of muscle atrophy/wasting.-   38. A method of inducing or stimulating myogenic differentiation of    isolated mesencymal stem cells in vitro comprising, contacting the    mesencymal stem cells with an effective amount of a retinoic acid    receptor gamma (RARγ) agonist.-   39. The method of paragraph 38, wherein contacting is for a time    period selected from the group consisting of about 12 hours, about 1    day, about 2 days, and about 3 days.-   40. A method of inducing or stimulating lineage-directed    differentiation of a pluripotent stem cell into a mesenchymal    lineage, the method comprising contacting the pluripotent stem cell    with an effective amount of a retinoic acid receptor gamma (RARγ)    agonist.-   41. The method paragraph 40, wherein the pluripotent stem cell is an    induced pluripotent stem cell.-   42. The method any one of paragraphs 40-41, wherein the pluripotent    stem cell is a mesenchymal stem cell.-   43. The method of any one of paragraphs 40-42, wherein the    mesenchymal lineage is selected from the group consisting of    myogenic, osteogenic, chodrogenic, tendonogenic, ligamentogenic,    maroow stromagenic, adipogenic, and dermogenic.-   44. The method of any one of paragraphs 40-43, wherein the    pluripotent stem cell is mammalian.-   45. The method of any one of paragraphs 40-44, wherein the    pluripotent stem cell is rodent.-   46. The method of any one of paragraphs 40-45, wherein the    pluripotent stem cell is human.-   47. The method of any one of paragraphs 40-46, wherein the RARγ    agonist is selected from the group consisting of CD-271    (6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic    acid); CD-394, CD-437    (6-(4-Hydroxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic    acid); CD-1530    (4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoic    acid); CD-2247; palovarotene    (4-[(1E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]-benzoic    acid); BMS-270394    (3-Fluoro-4-[(R)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic    acid); BMS-189961    (3-Fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic    acid); CH-55    (4-[(E)-3-(3,5-Di-tert-butyl-phenyl)-3-oxo-propenyl]-benzoic acid);    6-[3-(adamantan-1-yl)-4-(prop-2-ynyloxy)phenyl]naphthalene-2-carboxylic    acid;    5-[(E)-3-oxo-3-(5,5,8,8-tetrahydronaphthalene-2-yl)propenyl]thiophene-2-carboxylic    acid; and enantiomers, derivatives, prodrugs, and pharmaceutically    acceptable salts thereof.-   48. A composition comprising a mesenchymal stem cells wherein a    portion of the mesenchymal stem cells have been pretreated by    contact with a RAR agonist to thereby generate pretreated    mesenchymal stem cells.-   49. The composition of paragraph 43, wherein the portion of    pretreated mesenchymal stem cells is selected from the group    consisting of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%.-   50. A composition comprising a pluripotent stem cell population,    wherein at least one cell of the population was contacted with a    RARγ agonist to thereby generate a pretreated pluripotent stem cell.-   51. The composition of paragraph 50, wherein the pluripotent stem    cell is an induced pluripotent cell.-   52. The composition of any one of paragraphs 50-51, wherein the    pluripotent stem cell is a mesenchymal stem cell.-   53. The composition of any one of paragraphs 50-52, wherein the stem    cell is an isolated stem cell.-   54. The composition of any one of paragraphs 50-53, wherein the stem    cell is mammalian.-   55. The composition of any one of paragraphs 50-54, wherein the stem    cell is murine.-   56. The composition of any one of paragraphs 50-54, wherein the stem    cell is human.-   57. The composition of any one of paragraphs 50-56, wherein the RARγ    agonist is selected from the group consisting of CD-271    (6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic    acid); CD-394, CD-437    (6-(4-Hydroxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic    acid); CD-1530    (4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoic    acid); CD-2247; palovarotene    (4-[(1E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]-benzoic    acid); BMS-270394    (3-Fluoro-4-[(R)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic    acid); BMS-189961    (3-Fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic    acid); CH-55    (4-[(E)-3-(3,5-Di-tert-butyl-phenyl)-3-oxo-propenyl]-benzoic acid);    6-[3-(adamantan-1-yl)-4-(prop-2-ynyloxy)phenyl]naphthalene-2-carboxylic    acid;    5-[(E)-3-oxo-3-(5,5,8,8-tetrahydronaphthalene-2-yl)propenyl]thiophene-2-carboxylic    acid; and enantiomers, derivatives, prodrugs, and pharmaceutically    acceptable salts thereof.-   58. A pharmaceutical composition comprising a composition of any one    of paragraphs 50-57 and a pharmaceutically acceptable carrier.-   59. A kit for repairing or regenerating muscle, comprising at least    one of    -   (i) a RARγ agonist;    -   (ii) a RARγ agonist and a stem cell; or    -   (iii) a composition of any one of paragraphs 48-58.-   60. The kit of paragraph 59, wherein the stem cell is an induced    pluripotent stem cell.-   61. The kit of any one of paragraphs 59-60, wherein the stem cell is    a mesenchymal stem cell.-   62. The kit of any one of paragraphs 59-61, wherein the RARγ agonist    is formulated in a pharmaceutical composition.-   63. The kit of any one of paragraphs 59-62, wherein the RARγ agonist    is formulated for topical application.-   64. The kit of any one of paragraphs 59-63, wherein the stem cell is    an isolated stem cell.-   65. The kit of any one of paragraphs 59-64, wherein the stem cell is    mammalian.-   66. The kit of any one of paragraphs 59-65, wherein the stem cell is    rodent.-   67. The kit of any one of paragraphs 59-65, wherein the stem cell is    human.-   68. The kit of any one of paragraphs 59-67, wherein the RARγ agonist    is selected from the group consisting of CD-271    (6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic    acid); CD-394, CD-437    (6-(4-Hydroxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic    acid); CD-1530    (4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoic    acid); CD-2247; palovarotene    (4-[(1E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]-benzoic    acid); BMS-270394    (3-Fluoro-4-[(R)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic    acid); BMS-189961    (3-Fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic    acid); CH-55    (4-[(E)-3-(3,5-Di-tert-butyl-phenyl)-3-oxo-propenyl]-benzoic acid);    6-[3-(adamantan-1-yl)-4-(prop-2-ynyloxy)phenyl]naphthalene-2-carboxylic    acid;    5-[(E)-3-oxo-3-(5,5,8,8-tetrahydronaphthalene-2-yl)propenyl]thiophene-2-carboxylic    acid; and enantiomers, derivatives, prodrugs, and pharmaceutically    acceptable salts thereof.-   69. The kit of any one of paragraphs 59-68, further comprising    instructions for use.

The present invention is further illustrated by the following Examples.These Examples are provided to aid in the understanding of the inventionand are not construed as a limitation thereof.

EXAMPLES Example 1

Methods

Isolation and Culture of Bone Marrow Derived Mesenchymal Stem Cells(MSCs).

MSCs were isolated as described previously (ref) with minormodification. Briefly, mouse bone marrow cells (1-2×10⁷) harvested fromlong bones of 4-6 weeks old mice were seeded onto 100-mm culture dishes,incubated for 3 hours at 37° C. to allow attachment of adherent cells,and then rinsed twice with PBS to remove the nonadherent cells. The bonemarrow-derived MSCs formed adherent colonies after 12-15 days ofculture. Primary cultures were passed to disperse the colony-formingcells (passage 1). The cells were then subcultured again when theyreached 70% confluent. Cells were maintained in α-MEM (Gibco BRL;Invitrogen Corp.) containing 20% fetal bovine serum (FBS; Equitech-BioInc.), 2 mM L-glutamine, a combination of 100 U/ml penicillin and 100μg/ml streptomycin (Biofluids Inc.), and 55 μM 2-mercaptoethanol (GibcoBRL; Invitrogen Corp.) before the first passage. After the firstpassage, cells were maintained in 10% FBS, α-MEM unless indicated.

DsRED expressing MSCs were isolated from B6.Cg-Tg(CAG-DsRed*MST)1Nagy/Jmice (Jax Mice). GFP expressing MSCs were obtained from the transgenicmice carrying H2K-GFP (Dominici et al. Genesis 42:17-22 (2005)).

Cell Transplantation.

MSCs (1×10⁵) with or without RAR agonist pretreatment were suspended in10 μl serum-free DMEM and placed onto the muscle defect created asdescribed herein.

Histological Procedures.

To inspect muscle tissue structure, samples were fixed in 4% PFA andembedded in paraffin. Five μm serial sections were stained by either H&E(http://www.ihcworld.com/_protocols/special_stains/HE_Mayer.htm) orMasson's trichrome staining solution(http://www.ihcworld.com/_protocols/special_stains/masson_trichrome.htm).To detect adipose tissue, 4% PFA fixed samples were equilibrated in 20%sucrose in PBS, embedded in OCT compound and then sectioned by cryostat.Ten μm cryosections were dried, rehydrated and then stained with Oil-redO (http://www.ihcworld.com/_protocols/special_stains/oil_red_o.htm).

Immuno-Fluorescence.

For the detection of various antigens in tissue sections, 5 μm paraffinsections were de-paraffinized, re-hydrated and then treated with 0.1%pepsin in 0.02 N HCl for 15 min at 37° C. Sections were washed threetimes in PBS-T and blocked in 10% normal goat serum with 0.3% BSA.Sections were incubated with 1st antibody, washed three times with PBS-Tand then incubated with 2^(nd) antibody. The list of antigens,antibodies and their dilutions are shown below.

MyoD:Anti-MyoD; sc-760 (Santa Curz, 1:100), Anti-Rabbit Alexa Flour 488(Invitrogen, 1:500)

Myf5: Anti-Myf5; sc-302 (Santa Curz, 1:100), Anti-Rabbit Alexa Flour 488(Invitrogen, 1:500)

Anti-Laminina2; ALX-804-190 (Enzo Life Science, 1:250), Anti-Rat AlexaFlour 594 (Invitrogen, 1:500)

Myosin heavy chain (MHC): Anti-MHC: MF-20 (Developmental StudiesHybridoma Bank, 1:25), Anti-mouse Alexa Flour 588 (Invitrogen, 1:500)

Osteocalcin (OC): Anti-OC; M173 (Takara, 1:500), Anti-Rabbit Alexa Flour594 (Invitrogen, 1:500)

Green fluorescent protein (GFP): Biotin conjugated Anti-GFP; NB100-1678(Novus, 1:250), Streptavidin Alexa Flour 488 (Invitrogen, 1:500)

Local and Systemic Administration of RAR Agonist.

Systemic administration of RAR agonist was carried out by gavage asdescribed previously (Shimono et al. J. Orthop. Res. 28:271-277, 2010).Local delivery of RAR agonist was done by injecting RAR agonist(dissolved in DMSO) using Hamilton syringe 87943 with 26 G needle.

Results

The differential effects of retinoid agonists on myogenicdifferentiation in mouse bone marrow-derived mesenchymal stem cell(BMSC) cultures were investigated. BMSCs were prepared from femurs andtibias of 6 week-old mice by standard methods and maintained in 5% FBSDMEM for 7 days in presence of 0.1% DMSO (vehicle: Control), 1 μMall-trans retinoic acid (RA) (commercially available from Sigma), 1 μMRAR alpha-agonist (obtained from NuRx Pharmaceuticals) or 30 nM RARgamma-agonist (CD1530, commercially available). Many elongatedmultinucleated muscle cells formed only in RAR gamma-agonist treatedcultures.

Cells from a mesenchymal stem cell line were cultured for 10 days in thepresence or absence of RAR gamma-agonist (CD1530, 100 nM). Cultures wereprocessed for immunostaining with MyoD or Myf5 antibodies or stainedwith nuclear dye DAPI. Control untreated cells contained undetectableamounts of MyoD and Myf5, but both proteins were clearly present ingamma-agonist treated cultures. Results indicated an increase in MyoDand Myf5 levels by the RAR gamma-agonist in mesenchymal stem cell (MSC)cultures.

A muscle defect mouse model was used to investigate the muscle repairstimulation by administration of RAR gamma-agonists. A round-shapeddefect was created with an electric cautery in calf or anterior tibialmuscles in 8 week-old mice resulting in a 2.0 mm×2.0 mm×2.0 mm defect.

Round-shaped defects were created in calf-muscle tissue of 8 weeks oldmice as above (one muscle defect/mouse). Mice received corn-oil(vehicle) or 300 μg RAR gamma agonist (CD1530)/day by gavage on day 8,10 and 12 after injury. Tissue was collected for histological analysison day 14. Muscle defects in control mice were largely filled withscar-like fibrous and connective tissues by day 14. These scar-liketissues were totally negative for myosin heavy chain (MHC) as detectedby immunostaining. In contrast, the muscle defect sites in RARgamma-agonist treated mice were filled with many elongated muscle-likecells and many of these cells were MHC positive. Results indicatestimulation of muscle repair by systemic administration of RARgamma-agonist.

Following creation of 2×2×2 mm defect in the center of anterior tibialmuscle as above (one muscle defect/mouse), mice received corn-oil(vehicle) or 300 μg RAR gamma agonist/day by gavage on day 8, 10 and 12after injury. Tissues were collected for histological analysis at 2 and4 weeks. Macro-photos of injured muscle tissue collected from controland RAR gamma-agonist (CD1530)-treated mice were taken at 4 weeks afterinjury. In controls, the defect was clearly visible as a white spot. Incontrast, there was almost no visible sign of the original defect in theRAR gamma-agonist treated mice. Histological analysis revealed that themuscle tissue defects in control mice were occupied by mixtures ofadipose and fibrous tissue at 2 and 4 weeks. Deposition of fibroustissue was also noted between muscle fibers. However, in the RARgamma-agonist treated mice, the muscle defects were filled with musclefibers aligned along the major longitudinal axis of the anterior tibialmuscle. Results indicated full repair of muscle defects by systemicadministration of RAR gamma-agonist over time.

Histological analysis of skeletal muscle tissue was performed 4 weeksafter injury. Serial transverse sections of muscle tissue specimenscollected 4 weeks after injury (same samples as discussed directlyabove) were stained with Masson's trichrome or processed forimmuno-staining with antibodies to laminin (an extracellular proteinabundant around muscle cells). In controls, the initial injury site waspoorly repaired and largely occupied by adipose tissue and fibrousconnective tissue that did not stain with anti-laminin antibodies. Incontrast, the muscle injury was almost completely repaired ingamma-agonist treated animals; the repair tissue stained strongly withanti-laminin antibodies. To evaluate and quantify the changes ofskeletal muscle tissue structure, images of trichrome-stained serialsections were taken and the relative amounts of muscle fiber, adiposeand fibrous tissues were determined in multiple defined areas byImagePro software. The analyzed areas were 9×9 grids each (about 3×3 mm)that included the original injured site (2×2 mm). Muscle injury caused adecrease in total muscle fiber area and a concurrent increase in fibrousand adipose tissues area. In contrast, CD1530 treatment largely restoredthe tissue composition. The relative amount of muscle as determined isshown graphically in FIG. 1.

To verify that the RARgamma is required for muscle repair andregeneration, muscle injuries were created in wildtype (WT) andRARgamma-null mice as above. The animals were then treated withgamma-agonists or vehicle (corn oil). Tissue samples were collected 4weeks after injury, sectioned and stained with Masson's trichromestaining solution. In agreement with the above discussed results,gamma-agonist treatment induced effective muscle repair in WT mice.However, it had minimal effects in RARgamma-null mice in which themuscle defect site was filled with adipose and some connective tissuecells and lined with fibrous cells. These findings indicate that: (1)the muscle repair effects by the gamma-agonists are mediated byRARgamma; and (2) RARgamma is required for repair of skeletal muscletissue.

The effects of various retinoid agonists on skeletal muscle repair wereexamined. Macro photographs were taken of muscle injury sites at the4-week time point. Histo-morphometric analysis of composition of injuredmuscles is shown in FIG. 2. Muscle injury defects were created in 8week-old mice as described above. Mice received corn oil (control), 300μg pan-agonist retinoic acid (RA), 900 μg NRX195183 (RARalpha agonist)or 300 μg CD1530 (RARgamma agonist) on day 8, 10 and 12 by gavage. Fourweeks after injury, tissue samples were collected, processed forhistological analysis and subjected to histomorphometric quantification.In control animals and RARalpha-agonist- or RA-treated animals, theinjury sites were still clearly visible as large whitish spots due tothe replacement of muscle with adipose tissue. However, in theRARgamma-treated animals, the injury sites were essentially invisible at4 weeks since they had been filled with repair muscle tissue. Toquantify the responses, serial sections of tissues at the repair sitewere stained with trichrome and analyzed for quantification of relativeamounts of muscle, adipose and fibrous tissues in defined areas byImagePro software as described above. The analyzed areas were 9×9 gridseach (about 3×3 mm) that contained the original injury site (2×2 mm).Results are shown in FIG. 2. The graph shows averages of nine sectionsfrom three mice. “Normal” was derived from a muscle tissue compositionof uninjured normal muscle. As to be expected, muscles in controlanimals were composed of 90% muscle tissue, 7% fibrous tissue and traceamount of fat tissue. The muscle injury caused a dramatic decrease inmuscle fiber area from 90% to 33%, and there were correspondingincreases in adipose and fibrous tissue areas. Treatment with RAmoderately improved muscle fiber area from 33% (control) to 54%, butthere was a significant increase of fibrous tissue area. The RAR alphaagonist NRX195183 did not improve tissue composition. In contrast, thecomposition of repair tissues in CD1530-treated animals was almostnormal.

The effects of RARgamma agonists with different backbone structure onmuscle repair was investigated. In the experiments discussed above, thegamma-agonist CD1530 was shown to dramatically accelerate muscle repairand that RARgamma expression is required for such effect. To furthervalidate these findings and identify possible more potentgamma-agonists, three other structurally different selective RARgammaagonists (BMS270394, Palovarotene. and CD437) were tested in musclerepair. Leg muscle injuries were created as above. Each group of micereceived one of the different agonists (100 μg/gavage/day) or vehicle atday 8, 10 and 12 from injury. Tissues were collected 4 weeks aftersurgery and examined as above. In controls, the muscle injury sites wereclearly visible as whitish spots and there was minimal muscle repair; inaddition, fibrous scar tissue was present within the injured muscle. Incontrast, each gamma-agonist induced effective muscle repair; among thethree agonists tested, BMS270394 was the most effective and completelyrestored to normal muscle structure. The results indicate thatgamma-agonists are extremely effective in triggering muscle repair.

The effects of local administration of RARgamma agonist on muscle repairwere examined. Although major side effects of systemic administration ofgamma agonists have not been observed in the muscle repair experiments,it could be necessary to delivered them locally in certain clinicalsituations. To test the effectiveness of these drugs when given locally,muscle injuries were created as above and 3 μg of CD1530 was injectedsubcutaneously around the injury site on day 8 and 10 after operation.Vehicle was injected in controls, and tissues were collected at 4 weeksand examined as above. In controls, the injury sites were clearlyvisible and largely occupied by adipose tissue. In CD1530-treated group,however, the muscle defects were almost completely repaired. Themolecular weight of the gamma-agonists is in general less than 500 andtherefore, the drugs could be delivered from skin to deeper muscletissues in the form of ointments or other formulations (in addition tolocal injection).

GFP-expressing mouse mesenchymal stem cells were treated with vehicle(control) or 100 nM CD1530 for three days in culture, mixed withMatrigel and 1 μg rhBMP2, and then transplanted into nude mice. Twoweeks after implantation, ectopic tissue masses were collected foranalysis. Massive endochondral bone formation was observed in controls;anti-GFP and anti-OC (osteocalcin) immunostaining revealed that thetransplanted MSCs had contributed to bone formation. However, no ectopicendochondral bone had formed in mice transplanted withgamma-agonist-pretreated MSCs; these cells were present as indicated bypositive GFP staining but were negative for OC staining and were oftenaligned. Some cells were fused or in the process of fusion. Thus, thegamma agonist treatment effectively primes MSCs to undertake a myogenicdifferentiation pathway. These results indicate that RARgamma agonist isa priming factor for myogenic differentiation of mesenchymal stem cells(MSC).

To further examine the ability of gamma agonists to prime MSCs towardsthe myogenic lineage, muscle injury defects were created in nude mice asdiscussed above and DsRed-expressing MSCs pretreated with gamma-agonistfor three days (or left untreated) were transplanted thereto. Two weeksafter transplantation, tissues present at injury sites were collectedand inspected under fluorescent stereomicroscope. Tissues were thenprocessed for histological analyses. Fluorescent stereomicroscopyrevealed strong red fluorescence signal present in muscle injury siteswhere gamma-agonist-pretreated MSCs were implanted, but little to nofluorescence was observed in sites implanted with control untreated MSCs(A, left panels). Staining with oil red O revealed the presence ofabundant adipose tissue in injury sites implanted with control MSCs (B,left), but absence of these cells in the sites implanted withgamma-agonist-pretreated MSCs (B, right). Immunofluorescence analysisrevealed that DsRed-positive and gamma-agonist-pretreated MSCscontributed to the formation of MHC-expressing muscle repair tissue (C,right), but their absence in control. (DsRed, red color; MHC, green;DAPI, purple) In experiments described above, systemic administration ofgamma-agonists induced repair of muscle injury defects in 4 weeks. It isnoteworthy that similar defects were almost completely repaired within 2weeks after transplantation of gamma-agonist primed MSCs. Therefore theuse of gamma-agonist pretreated MSCs could represent a very effectiveprocedure to repair muscle injuries quickly. These results indicaterepair of muscle injury by gamma-agonist-primed MSCs.

Example 2

Methods

Unless otherwise indicated, all methods were performed as described inExample 1, or by standard procedures known in the art.

Immunodetection of human cells and myosin heavy chain was performed byincubating sections with biotinylated anti-human TRA-1-85 (1:250dilution, R&D cat #BAM3195) and anti-laminin (1:250, Enzy, ALX-804-190),washed and then incubated with Alexa Fluor 488streptavidine and AlexaFluor 594 anti-rat IgG.

Results

Repair of Muscle Injury by Gamma-Agonist-Pre-Treated Human AdiposeTissue Derived Mesenchymal Stem Cell.

The experience described below indicate that RAR γ agonist pre-treatedstem cells not only regenerate muscle tissue by themselves, but alsosupport myogenic differentiation of host cells. As such, these cells arenow referred to as “pre-treated” rather than “primed” mesenchymal stemcells, to be consistent with the accepted definition of the term “prime”as it is used in the art to mean to commit cells to undergo myogenesis.

To test the effects of pretreatment of an human mesenchymal stem cellwith RAR gamma-agonist, a round-shaped defect was created in theanterior tibial muscle of NOD mice (NOD.Cg-Rag1tm1Mom Prf1tm1Sdz/Sz) asdescribed above. Non-treated or RARg treated human adipose derivedmesenchymal stem cells were transplanted to the site (5,000 cells persite). Tissue was collected 2 weeks after operation and subjected tohistological analyses by Masson's trichrome staining sections of RARgpretreated MSC transplanted muscle and control MSC transplanted muscle,and also by immunol-fluorescence detection of myosin heavy chain (MHC,anti-human-derived cell antigen, and nucleus (DAPI) of RARg pretreatedMSC transplanted muscle and control MSC transplanted muscle.

RARg pretreated human MSC clearly facilitated repair of the damagedmuscle tissue. While RARg pretreated human MSCs contributed muscletissue regeneration, MSCs that were not pre-treated were mostly excludedfrom the muscle tissue.

To test if RAR gamma-agonist pre-treatment is effective on differenttype of mesenchymal stem cells, human adipose tissue derived mesenchymalstem cells (Zenbio, Cat # ASC-F) were treated with 1 μM CD1530 orvehicle for 3 days in vitro, and then transplanted into a muscle damagemodel system. A round-shaped defect was created in the anterior tibialmuscle of 6-week old NSG mice (NOD scid gamma mouse, Jax mice #005557)as described above (NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ). Non-treatedor RARg treated human adipose derived mesenchymal stem cells weretransplanted to the site (10,000 cells per site). Tissue was collected 2weeks after operation and subjected to histological analyses (Masson'strichrome staining sections of RARg primed MSC transplanted muscle andcontrol MSC transplanted muscle, and also by immuno-fluorescencedetection of laminin, human-derived cell antigen, and nucleus (DAPI) ofRARγ agonist pretreated MSC transplanted muscle and control MSCtransplanted muscle.

RARg pre-treated human MSC clearly facilitated repair of the damagedmuscle tissue. While RARγ pretreated human MSCs were observed to bemainly distributed around muscle fibers, non-treated cells were mostlyexcluded from the muscle tissue. These results indicate that the use ofRARγ agonist pretreated stem cells such as MSC is an effective therapyfor muscle repair and also that stem cells (e.g. MSC) from varioussources can be used for this therapy.

Repair of Complex Tissue (Muscle+Bone) Injury with Primed (RARg AgonistTreated) or Non-Primed MSCs

Sever muscle injury often accompanies damages of other tissue such asbone, tendon and nerve. By using a simplified compound injury model,RARγ agonist treated MSCs were examined for preferential targeting toinjured muscle tissue.

Compound (bone and muscle) tissue injury was created in 2 month old miceby cutting fibula and surrounding muscle tissue. Following this, a 1:1mixture of RARγ agonist pre-treated MSC and control non-pretreated MSCswere transplanted near the injury site. The MSC were prepared eitherfrom GFP (pre-treated) or DsRED (non-treated) expressing mice bonemarrow. The injured tissue were collected 1 week after surgery,sectioned and examined for distribution of pre-treated andnon-pretreated MSCs using anti-GFP and anti-DsRed antibodiesrespectively. Histological analysis with HE staining revealed damagedbone and muscle tissues were still under repair process. Strikingly,while the majority of primed MSCs were detected in muscle tissue, themajority of primed cells were always associated with bone tissue. Someof the DsRed expressing RARγ agonist pretreated MSCs were observed toform typical multinucleated skeletal muscle cells.

The RARγ agonist pre-treated and non-treated MSCs were observed totarget different tissue injury sites. The results indicate that RARγagonist pretreated MSCs selectively target injured muscle tissue. RARγagonist pretreated MSCs will be useful to repair damaged muscle tissuewhen other tissues are also damaged. It has previous been reported thatsystemic administration of RARγ agonist not only inhibits heterotopicossification but delays fracture healing (Shimono et al., NatureMedicine 17, 454-460 (2011)). In addition these results strongly suggestthat the combined transplantation of pre-treated and nontreated MSCs atan appropriate ratio is an effective therapy for compound tissue injury.

Requirement of RARγ Signaling in Muscle Repair

The above results indicate that muscle tissue defect is poorly repairedin RARγ null mice. To further confirm this finding, another widely usedmuscle degeneration model was used. 1 μg cardio-toxin was injected intoanterior tibial muscle tissue of wild type and RARγ null mice. Muscletissue was collected two weeks after cardio-toxin injection, sectionedand examined by Masson's trichrome staining. The cardio-toxin injectedmuscle tissue was seen to be mostly restored with immature muscle fibersby 2 weeks in the control wild type mice. In contrast, RAR null muscletissue was seen to be poorly repaired.

Up-Regulation of Local Retinoid Signaling after Muscle Injury

Anterior tibial muscle of the retinoic acid signaling reporter mice(RARE-LacZ mice) were cut or sham-operated. The muscle tissue wascollected at days 1, 4 or 7 and stained with X-gal. The reporteractivity was observed to be increased 4 days after injury, suggestingthe involvement of this signaling pathway in muscle tissue repair.

Total muscle tissue RNA was collected at 8 hours, 2 days and 4 days, andsubjected to RT-PCR for the analysis of RAR signaling related geneexpression. The expression of ALDH2, the late limiting enzyme thatproduces RA from its precursor was seen to be up-regulated 4 days afterinjury, returning back to normal expression levels by day 11. Incontrast, Cyp26b1 (Cyp26B), the enzyme that degrades RA was seen to betransiently up-regulated at day 2 after injury. The results are shown inFIGS. 4 and 5, and indicate that the local RA concentration decreasesfirst and then transiently increases after muscle injury.

Effect of the Treatment Timing on Muscle Tissue Repair

Muscle defects were created as described above. Mice were treated withRARg agonist (CD1530) at days 1-3 or days 5-7. Pictures show the HEstaining of the muscle tissues of 4 weeks after injury. Muscle defectsin untreated controls were largely replaced with fat and fibrous tissue.The Day 1-3 treatment with RARg agonist decreased adipose and fibrousscar tissue formation but left a partial defect. In contrast, the muscledefects were completely filled with newly generated muscle fibers in theDay 5-7 treatment group. Thus the treatment timing is very important forfaster and better muscle tissue repair. RARg signaling should beenhanced when endogenous retinoid signal is increased. The suggestedtreatment regimen is local or systemic administration of RARg agonist onday 5 and 7 after muscle injury. Further enhancement of accuratetreatment timing can be achieved by measuring local retinoic acidconcentration by LS/MS/MS.

Muscle defects were created as described above. Mice were given 100 ngRARγ agonist (CD1530) every other day during the period of days 1-5,days 5-9, and days 1-9. Day 1-5 group mice received CD1530 on day 1, 3and 5. Day 5-9 group received CD1530 on day 5, 7 and 9. Day 1-9 groupreceived CD1530 on day 1, 3, 5, 7 and 9. The muscles were then comparedto vehicle treated injured muscle as well as intact muscle (un-injuredmuscle) tissue. The HE staining of the muscle tissues at 4 weeks afterinjury was examined. Muscle defects in untreated control were observedto be largely replaced with fat and fibrous tissue (Vehicle). Both day1-5 or 1-9 treatment with RARγ agonist decreased adipose and fibrousscar tissue formation but left a large indentation. The quality and theamount of newly formed muscle tissue were best in the Day 5-9 treatmentgroup, which had a much reduced indentation.

Histo-Morphometric Analysis of the Injured Muscle Tissue

Serial tissue sections at the repair site were stained with Masson'strichrome and analyzed to quantify relative amounts of muscle, adiposeand fibrous tissues in defined areas by ImagePro software as described.Results are shown in FIG. 6. Image analysis confirmed that Day 5-9treated group muscle tissue contains the least amount of adipose andfibrous tissue among all injured groups. This indicates that thetreatment timing is very important for faster and better muscle tissuerepair and that more beneficial results are obtained when RARγ signalingis therapeutically enhanced (e.g., by administration of an RARγ agonist)at the time of increased endogenous retinoid signaling, and not before.

The invention claimed is:
 1. A method of treating a subject sufferingfrom a muscle damaging myopathy, comprising administering to the subjecta therapeutically effective amount of a retinoic acid receptor gamma(RARγ) agonist, wherein the RARγ agonist is4-[(E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]benzoicacid or a pharmaceutically acceptable salt thereof.
 2. The method ofclaim 1, wherein the muscle damaging myopathy is myositis ossificans. 3.The method of claim 1, wherein the muscle damaging myopathy is acongenital myopathy.
 4. The method of claim 1, wherein the muscledamaging myopathy is an acquired myopathy.
 5. The method of claim 1,wherein said RARγ agonist is administered locally.
 6. The method ofclaim 1, wherein said RARγ agonist is administered systemically.
 7. Amethod of inducing progenitor stem cells to undergo myogenicdifferentiation in a subject suffering from a muscle damaging myopathy,comprising administering to the subject a therapeutically effectiveamount of an RARγ agonist, wherein the RARγ agonist is4-[(E)-2-[5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-2-naphthalenyl]-ethenyl]benzoicacid or a pharmaceutically acceptable salt thereof.
 8. The method ofclaim 7, wherein the muscle damaging myopathy is myositis ossificans. 9.The method of claim 7, wherein the muscle damaging myopathy is acongenital myopathy.
 10. The method of claim 7, wherein the muscledamaging myopathy is an acquired myopathy.
 11. The method of claim 7,wherein said RARγ agonist is administered locally.
 12. The method ofclaim 7, wherein said RARγ agonist is administered systemically.